Systems and methods for targeted breast cancer therapies

ABSTRACT

Systems and methods for producing liposomes, including control liposomes and immunoliposomes targeting breast cancer are provided. Systems and methods for treating breast cancer, using targeted immunoliposomes produced according to various methods are also disclosed herein. For example, trastuzumab-conjugated immunoliposomes may be used to deliver chemotherapeutic agents to breast cancer tissues for the treatment of breast cancer. Systems and methods for actuating liposomes using ultrasound are also disclosed, such as systems and methods for actuating trastuzumab-conjugated liposomes accumulated in breast cancer tissues for the treatment of breast cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/572,358, filed on Oct. 13, 2017, which is hereby incorporated byreference in its entirety and should be considered a part of thisspecification.

BACKGROUND Field

The present application relates generally to systems and methods forproducing acoustically activated or triggered nanoparticles and morespecifically, relates to systems and methods for producing and usingacoustically activated or triggered, ligand-targeted liposomes for thenovel treatment of cancer.

Description of the Related Art

Cancer is a wide-spread disease, and the most common types (responsiblefor more than half of all cases) are breast, prostate, lung, and coloncancers. By far the most common cancer in women and the second leadingcause of cancer death among American women is breast cancer. It is alsoa global concern that endangers women's lives worldwide. Breast canceris the most frequent cancer in women and the second overall. Of the 4.4million new breast cancer cases diagnosed within the past five years,only 1.4 million have survived.

In recent years, there have been several ways to treat cancer dependingon the type of malignancy, stage, and pathologic features such asreceptor status and tumor grade. A plan for each patient depends on thepurpose of treatment, either to shrink the tumor, stop the tumor growth,or just enhance the patient's quality of life in late stages. Treatmentsinclude surgery, radiation, chemotherapy, biological therapy (includingimmunotherapy), and targeted therapy. It can also be a combination ofthe above treatments. In the early stages before the metastasis ofcancer, surgery combined with radiation (localized treatments) may beapplied. But in later stages, chemotherapy may be used, sometimes incombination with biological therapy, such as immunotherapy. Adjuvanttreatment may be then followed to make sure that new tumors areeliminated.

SUMMARY

In some embodiments, a method of treating breast cancer in a mammalcomprises: delivering an actively targeted liposome to the mammal,allowing the actively targeted liposome to circulate throughout acirculatory system of the mammal for a time sufficient to allowaggregation of a therapeutic quantity of actively targeted liposomes ata treatment area comprising a breast cancer; and applying ultrasound tothe treatment area such that the actively targeted liposome iscritically disrupted thereby releasing the chemotherapeutic drug in thetreatment area. The actively targeted liposome comprises: a lipidbilayer forming a spherical shell comprising an interior liposomalcavity; a plurality of trastuzumab molecules linked to a surface of theactively targeted liposome; and a chemotherapeutic drug comprising atleast one of a hydrophilic chemotherapeutic drug contained within theinterior liposomal cavity and a hydrophobic chemotherapeutic drugcontained within the lipid bilayer of the actively targeted liposome.

The ultrasound applied to the treatment area may comprise a lowfrequency ultrasound. The low frequency ultrasound may comprise a 20 kHzwith a power density of one of 7.46 W/cm2, 9.85 W/cm2, and 17.31 W/cm2.The low frequency ultrasound applied to the treatment area may beapplied for less than about 6 minutes. The ultrasound applied to thetreatment area may comprise high frequency ultrasound. Pulsed ultrasoundmay be applied to the treatment area. The ultrasound may be pulsed 2times or 3 times. The lipid bilayer of the actively targeted liposomemay comprise one or more PEGylated lipids. The plurality of trastuzumabmolecules may be linked to a distal end of the PEG chain. The activelytargeted liposome may comprise a protein density of 7.5-30 molecules perliposome. The actively targeted liposome may comprise a mean radiusbetween 50-150 nm. The actively targeted liposome may comprise 6 to 12trastuzumab molecules. The chemotherapeutic drug may comprise calcein.The chemotherapeutic drug may be selected from the group of doxorubicin,annamycin, daunorubicin, vincristine, cisplatin derivatives, paclitaxel5-fluorouracil derivatives, camptothecin derivatives, and retinoids. Theactively targeted liposome may be comprises a plurality of vesicles, theplurality of vesicles linked to about 9 trastuzumab molecules. Theplurality of trastuzumab molecules may be linked to the surface of theactively targeted liposome using cyanuric chloride.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates a schematic view of molecule structure ofphosphoglyceride.

FIG. 2 illustrates a classification of liposomes.

FIGS. 3A-D illustrates embodiments of structures of liposomes andimmunoliposomes.

FIG. 4 illustrates an embodiment of protein levels for control liposomesand immunoliposomes.

FIG. 5 illustrates an embodiment of release profiles for NH₂ liposomestriggered by ultrasound.

FIG. 6 illustrates an embodiment of the cumulative fraction releasedmeasured at different pulses and the final plateau for NH₂ liposomes.

FIG. 7 illustrates an embodiment of fractional release measured atdifferent pulses for NH₂ liposomes.

FIG. 8 illustrates an embodiment of a release profile forimmunoliposomes triggered by ultrasound.

FIG. 9 illustrates an embodiment of cumulative fractions releasedmeasured at different pulses and the final plateau for immunoliposomes.

FIG. 10 illustrates an embodiment of cumulative fractions releasedmeasured at different pulses and the final plateau for immunoliposomes.

FIG. 11 illustrates an embodiment of a release profile for NH₂ liposomesand immunoliposomes at different power densities.

FIG. 12 illustrates an embodiment of fractional release after the firstpulse measured for NH₂ liposomes and immunoliposomes at each powerdensity.

FIG. 13 illustrates an embodiment of fractional release after the secondpulse measured for NH₂ liposomes and immunoliposomes at each powerdensity.

FIG. 14 illustrates an embodiment of the final cumulative fractionreleased for NH₂ liposomes and immunoliposomes at each power density.

FIGS. 15-23 illustrates an embodiment of comparison between releaseprofile of NH₂ Liposomes and different kinetic models.

FIGS. 24-26 illustrates an embodiment of parity plots for the average ofthe three batches of NH₂ liposomes at each power density, usingparameters estimated for different model fits.

FIGS. 27-29 illustrates an embodiment of parity plots for the average ofthe three batches of immunoliposomes at each power density, usingparameters estimated for different model fits.

DETAILED DESCRIPTION Cancer

Most normal cells replicate, but tumor cells are damaged cells thatcannot stop growing, such that they can escape programmed cell death(apoptosis); due to abnormalities in cell proliferation,differentiation, and survival. These cells are either benign ormalignant. Malignant cells (cancer) usually proliferate faster andspread throughout the body invading other organs. Solid tumors that stemfrom cells of mesenchymal origin (connective tissue) are known assarcomas, and they spread via the blood stream. Cancer cells that arisefrom epithelial cells are called carcinomas, they are the most commontype of human cancer, and they spread through the lymphatic channels.Leukemia and lymphomas arise from the cells of the blood and the immunesystem respectively.

The tumor starts from a single cell that mutates. Mutations includeactivation of oncogenes that promote cell proliferation and damage intumor suppressor genes that causes cell failure to differentiatenormally. It proliferates fast requiring extensive blood vessels andnutrients during proliferation. After that, these cells form a cluster,and in that stage, it is called in situ carcinoma. Then they penetratethe basement membrane (extracellular matrix of tissue) to invade theunderlying connective tissue. Once this happens, cancer cells cancirculate throughout the body and spread the tumor to other organs, in aprocess called metastasis, and cause metastatic cancers. Metastasis isthe main reason why localized therapy eventually does not work in cancertreatment. Furthermore, dissolution of the basement membrane tissuemakes it permeable and easy to penetrate, which is considered acharacteristic of the tumor tissue.

Apoptosis contributes to the development of the tumor and also createsresistance to chemotherapeutic drugs that are based on damaging the DNA.Normal cells proliferate in the presence of growth factors, thiscondition is determined by the density of the tissue, but cancer cellsare insensitive to such signals; which can be due to the unregulatedactivity of growth factors receptors and intracellular signalingsystems. Therefore, in general, cancer cells have a reduced requirementfor growth factors. Some cancer cells produce their own growth factors,auto-stimulating cell division (autocrine signaling). Cancer cells areless responsive to cell-cell interactions, so there is less adhesion toother cells which makes them rounder. This also plays a role in theirdisordered multilayered growth patterns in which they push neighboringcells to grow, and eventually put pressure on the organ. They secretechemicals that digest extracellular matrix components allowing for moreinvasions. They also produce growth factors that stimulate angiogenesiswhich results in the formation of new capillaries around the tumor. Itcan also help in metastasis since these capillaries are easy topenetrate by cancer cells. Divided cells lose the capacity todifferentiate. Cancer cells fail to respond to signals promotingapoptosis, contributing to the survival of damaged mutated cells. Somecancer cells produce immunosuppressive factors in an attempt to avoiddetection by the immune system. Lower pH around the tumor is caused bylactic acid formation due to the lack of oxygen which is a result of ahigher oxygen consumption rate in proliferation (higher than whatangiogenesis can provide). This also contributes to immune systemevasion where high lactic levels disturb T cells function.

Cancer progresses through a series of abnormalities that accumulate overtime, so there can be several factors that induce cancer includingcarcinogens. Carcinogens include environmental factors, chemicals,radiations, and viruses. Factors that contribute to the proliferation ofthe mutated cells are called tumor promoters, they include increasedaccumulation of hormones and/or collagen.

HER2 Overexpression

Breast cancer is the most frequent cancer in women and the secondoverall. Breast cancer most commonly spreads to the regional lymphnodes, and in advanced stages, it could also spread to the bones, lungs,and liver. Some factors that may increase the risk of breast cancer areearly menarche, late menopause, as well as obesity and increased uptakeof alcohol. Breast cancer is known to develop as a result of a series ofchanges in oncogenes and cell mutations that include mutations in BRAC1and BRAC2 genes. These two genes are tumor suppressor genes that helprepair DNA damage. Mutations of these genes are present in basal breastcancers (triple negative breast cancer i.e. those that are HER2negative, ER-negative and PR negative), and they may account for 20-25%of heredity breast cancers and 5-10% of all breast cancers.

Local estrogen production is also known to facilitate the growth of bothcancer cells and breast stromal cells. Some breast cancer cells,sometimes referred to as “hormone receptor positive” breast cancers,differentially express estrogen and progesterone receptors compared tonormal breast tissue. This type of cancer occurs most commonly in olderpatients and progresses slowly, and is expressed in up to 60% of breasttumors and can be treated with tamoxifen (an estrogen antagonist).Vascular endothelial growth factor receptors (VEGFR) are overexpressedin most cancer cells including breast cancer. They are essential forangiogenesis which is in turn required for cancer growth, invasion, andmetastasis. Insulin-like growth factor receptors are also expressed inhigh levels in breast cancer compared with normal breast tissue. Lastly,another important family of receptors that regulate cell proliferationand apoptosis are the human epidermal growth factors family thatincludes HER1 (EGFR), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4) thatbelong to a family of transmembrane receptor tyrosine kinases (RTKs).

HER2 encodes 1225 amino acids, and it is normally expressed at lowlevels in the epithelial cells of various organs such as the lung,bladder, pancreas, breast, and prostate. HER2 overexpression occurs in20% to 30% of patients with breast cancer, and it is more spread amongyounger woman. HER-2 positive cancer progresses rapidly; hence coincideswith decreased survival rates, with 20% less survival rate for HER-2positive women than a HER-2 negative woman in the five year periodfollowing surgery. It is noteworthy that HER2 overexpression in breastcancer cells corresponds to overexpression of VEGF, and thus inducingangiogenesis. HER2 does not have a known ligand (orphan receptor) but itheterodimers with other receptors in the family to enhance signaling.Overexpression or gene amplification of HER2 induces ligand-independenthomodimerization, hence the activation of HER2 signaling pathways thatinclude PI3K/AKT and RAS/RAF/MAPK pathways which lead to cellproliferation, growth and survival as well as invasion and angiogenesis,and therefore cancer development. The HER family of receptors bind to avariety of different ligands as shown Table 1. It was found thatprolidase (peptidase D) may act as a ligand for HER2 and inhibitdownstream signaling using immunoprecipitation. Additionally, mutationsin the PI3KCA gene have been detected in 25% of breast cancers. Table 1also shows that HER-2 positive tumors tend to include P53 tumorsuppressor gene mutation.

TABLE 1 Ligands of the human epidermal growth factors (HER) family HER1Ligand (EGFR) HER2 HER3 HER4 Transforming yes — — — Growth Factor alpha(TGFα) Amphiregulin (AR, yes — — — AREG) Epidermal Growth yes — — —Factor (EGF) Betacellulin (BTC) yes — — yes Heparin-binding yes — — yesEGF like Growth Factor (HBEGF) Epigen (EPG) yes — — — Epiregulin (EPR)yes — — yes Heregulin — — 1, 2 1, 2, 3, 4

Treatments of Cancer

In recent years, there have been several ways to treat cancer dependingon the type of malignancy, stage, and pathologic features such asreceptor status and tumor grade. A plan for each patient may depend onthe purpose of treatment, either to shrink the tumor, stop the tumorgrowth, or just enhance the patient's quality of life in late stages.Cancer treatments may include surgery, radiation, chemotherapy,biological therapy (includes immunotherapy), and targeted therapy. Itcan also be a combination of these treatments. In the early stagesbefore the metastasis of cancer, surgery combined with radiation(localized treatments) can be applied. In later stages, chemotherapyand/or immunotherapy may be used. Adjuvant treatment may be thenfollowed to make sure that tumors are eliminated. Sometimes chemotherapymay be used before surgery in order to shrink the tumor size.

In chemotherapy, drugs are used to inhibit cell division and/oreventually kill them. Nitrogen mustard was the first chemotherapeuticagent to be used against cancer. It targets fast proliferating cellswhich unfortunately include some other normal cells as well (i.e. stemscells). Folic acid antagonists are the second group developed and areused to inhibit the cancer cell ability to produce folic acid which maybe necessary for growth. Nucleic acid antagonists are the third groupused. They are used to inhibit nucleic acid which is also needed forcell growth and proliferation. Tyrosine kinase inhibitors work bydeactivating this protein which is responsible for facilitatingsignaling pathways related to cell proliferation activities. Andantitumor antibiotics such as doxorubicin are also used in cancertreatment.

In some instances, the challenge of using chemotherapy may reside withinthe non-selective action resulting in the severe side effects of thesechemical agents to normal cells which limit the dosage given in therapy.These side effects may include nausea, vomiting, diarrhea, anemia, andhair loss. They can also cause damage to the heart, the kidneys, and thebone marrow and may result in the death of the patient in some cases.

Another challenge can be the ability of the tumor to develop resistanceto the antineoplastic drug, rendering them ineffective in the fightagainst malignant tissues. In drug resistance (also known as multi-drugresistance-MDR), the cancer cells may develop a mechanism that reducesthe ability of cancer cells to take up the chemical agents, or reduceexpression of some proteins that guide the agent to the cell. Anadditional MDR mechanism may be developed when the tumor is grown andsome cells slow their proliferation, and hence become non-detectable asmalignant cells. The heterogeneous tumor consists of cells withdifferent characteristics and different sensitivity to chemotherapy;therefore they become drug resistant. As most chemotherapy drugs inducecancer cells apoptosis, defective apoptosis allows the survival of thesecells, and make them resistant in the process.

To overcome MDR, a combination of drugs is generally used. MDR can betreated by the administration of a combination of drugs that followdifferent cytotoxic mechanisms, increasing the drug dosage, usingchemotherapy with a combination of other therapies (immunotherapy),hyperthermia therapy, or delivering the drug more efficiently usingadvanced drug delivery systems. Hyperthermia therapy works by heatingthe tumor region above 40° C., a temperature at which cancer cells andtissue are rendered more porous, hence increases the uptake of the drug.It also facilitates the delivery of the drug as a result of dilatedblood vessels to the tumor. Hyperthermia therapy can also be used incombination with radiation therapy.

In some instances, biological therapy may be used, alone or incombination with other therapies to treat cancer. Biological therapyincludes: inducing the host defense (immunotherapy), inhibiting tumorgrowth, and/or prompting cell differentiation (remission). Celldifferentiation is coupled with cell division, in such a way thatwhenever a cell is matured and fully differentiated it stopsreplicating/dividing. Inhibiting tumor growth may work by inhibitingangiogenesis that provides nutrients needed for proliferation. This canbe accomplished by blocking growth factors receptors signals to preventtumor development.

Generally, the immune system recognizes pathogenic infections andabnormal or malfunctioning cells and destroys them. There are severaltypes of immunotherapy including adoptive, passive and activeimmunotherapy. Adoptive immunotherapy is based on transferring whiteblood cells into the host. First tumor-sensitive white blood cells(cytotoxic T-cells) may be taken from the tumor area and grown in thelaboratory to a large number, and then activated against thetumor-associated antigen (TAA) and injected back to help the host immunesystem fight cancer. The transplantation of the bone marrow could alsobe considered as adoptive immunotherapy. Non-specific activeimmunotherapy aims to boost the immune system in general by stimulatingmacrophages, lymphocytes, and natural killer cells.

In active immunotherapy, cells of a specific tumor can be altered to bemore antigenic (provoking to the immune system) to help identify thetumor and produce designed antibodies for that tumor. This has led tothe development of hybrid white blood cells that are cultured inlaboratories to be used as factories for producing one specific type ofantibodies (monoclonal antibodies).

Passive immunotherapy takes advantage of the development of monoclonalantibodies (mAb) and uses it to produce large quantities of monoclonalantibodies and transferring them into patients. Antibodies are proteinsthat bind to antigens expressed on the cancer cell surface marking themfor the destruction by macrophages. Some monoclonal antibodies workmainly by attaching to and blocking antigens on cancer cells. Theseantigens help cancer cells grow and spread. One example is the use oftrastuzumab (Herceptin) in blocking human epidermal receptor two protein(HER2), a receptor extensively overexpressed in breast cancer cells.Another example is the use of bevacizumab in targeting vascularendothelial growth factor VEGF receptor (that is necessary forangiogenesis), and cetuximab that binds the epidermal growth factorreceptor (EGFR or HER-1).

Some monoclonal antibodies are also used in blocking immune checkpointscreated by cancer cells in an attempt to avoid detection by the immunesystem. This works by binding the antibody to the cytotoxic T-lymphocyteassociated antigen (CTLA4) receptor on the T-cells of the immune system.The CTLA4 receptor functions are opposite to the CD28 receptor thatactivates the attack against cancer cells. These receptors work onantigens CD80 and CD86 expressed on the cancer cell. Other ligandsnormally found in cancer cells are programmed death ligand 1 (PD-L1)that binds to a PD-1 checkpoint protein on T-cells and prevent them fromattacking the cancer cell.

Targeted Therapy

One way to achieve more selective cancer treatment is the development ofdrugs that target specific receptors expressed on the surface of cancercells. Normally targets include receptors that play a role in cellgrowth and survival, and that are overexpressed only or mostly on cancercells rather than healthy ones. Targets can also include fusion genesthat result from abnormalities in chromosomes.

Receptors are proteins that help cells regulate their processes. Surfacereceptors are proteins that span the cell membrane and are exposed fromboth sides. The extracellular domain, the transmembrane protein, and theintracellular domain transfer signals through initiating chain reactionsusing other intracellular proteins to form intracellular pathways to thetarget inside the cell, usually the nucleus. Intracellular receptors areproteins located in the cytosol or nucleus. Receptors that mediate cellinteractions are called cell adhesion molecules in which they include:the selectins, the integrins, the immunoglobulin (Ig) superfamily, andthe cadherins. Each receptor may be activated by binding to its specificligand. Ligands are signaling molecules that transmit informationbetween cells. They include ions, hormones, neurotransmitters, peptides,and growth factors. Targeted therapy can work by interfering with thefunctions of targets, using monoclonal antibodies for surface targets(transmembrane receptors), and small molecules for intracellular targets(usually enzymes proteins or transcription factors).

In contrast to chemotherapy that works by killing cells as a result ofcytotoxic effects, targeted therapy is based on cytostatic effects(blocking proliferation). Monoclonal antibodies can also be used asconjugates guiding chemotherapy agents to the cell, or withradio-isotopes to focus radiotherapy on cancer cells.

There may be some challenges in implementing targeted therapy. Thechallenge may lie within identifying the target and what kind of ligandsdoes it respond to, and developing an agent that interacts solely withthat target. Developing resistance in which the cell mutates and stopsresponding to the drug can be another challenge. Another major challengeis related to monoclonal antibodies; in which creating humanizedmonoclonal antibodies is still a challenge but necessary because humanmouse monoclonal antibodies can sometimes induce allergic reactions.

To overcome the challenges in cancer treatment (discussed in theprevious sections) including severe side effects, and limited dosage anddrug resistance, developing new strategies of drug delivery such as drugdelivery systems may be needed. Drug delivery systems may be enable theusage of higher doses and to target cancer cells effectively withoutharming the healthy surrounding ones.

Drug Delivery Systems

Drug Delivery systems control the rate at which a drug is released andthe location in the body where it is released, hence subsequentlycontrolling the therapeutic agent infusion rate and required tissue druglevels. Some systems can control both. They not only improve safety andefficacy, but also permit new therapies that once were considered toorisky or toxic to deliver by conventional ways. Release patterns canaffect the therapeutic response significantly. Additionally, it can bemore economical to enhance drug delivery systems than it would be totreat the side effects associated with the conventional chemotherapy.Drug Delivery Systems can include: mechanical pumps (implants), polymermatrices (micro-particulates), externally applied transdermal patches,drug delivery vehicles, and/or combination thereof.

Nanoparticles such as liposomes and macromolecular drug carriers such aspolymers are classified as nanomedicines/nanocarriers, a fieldencompassing nanoscale drug delivery devices and aiming at optimizingselectivity, prolonging the agent's activity and controlling drugrelease and cellular uptake. The advantages of this technology are itsability to cross physiologic barriers, overcome drug resistance, andsignificantly reduce side effects. The small size of nanocarriers mayhelp them leave the vascular system and extravasate at the tumor sites.It has also raised the possibility for intracellular targeting and genedelivery.

In embodiments, nanocarriers have the ability to passively target tumorcells by utilizing the enhanced permeability and retention (EPR) effect,which are discussed further in details elsewhere in this specification.Additionally, nanocarriers can be actively targeted utilizing theligand-receptor by attaching different moieties on their surface. As aresult, they have increased selectivity for tumors in general, and canmake tumor specifically targeted. After reaching the tumor site,nanocarriers can be triggered to release their contents faster.

Triggering nanocarriers internally can be achieved via severalstimulators such as change in pH, temperature, pressure, or enzymesconcentration. Otherwise, nanocarriers can be triggered externally bylight, magnetic or electric fields, and ultrasound.

In embodiments, nanocarriers may have a size range of about: 10-800 nm,200-600 nm, 300-500 nm, or 400 nm. In some embodiments, nanocarriersinclude nanocrystals (quantum dots), nanosuspensions, nanotubes,nanowires, micelles, liposomes, metal-organic frameworks (MOFs), ceramicnanoparticles, dendrimers, solid lipid nanoparticles, and hydrogelnanoparticles. Micelles and liposomes are the most widely applicablenanocarriers.

Dendrimers are highly branched mono-dispersed macromolecules with asymmetric structure that may range in size from about: 1 to 20 nm, 2-18nm, 3-15 nm, 4-12 nm, or 8-10 nm. Dendrimers contain nanocavities thatare well suited for drugs encapsulation, and terminal functional groupsthat determine their solubility and chemical activity. By choosingdifferent branching units and surface groups, properties of dendrimerscan be altered. The diversity of structural components that can be used,provide them with distinctive physical and chemical properties. Incontrast, toxicity and rapid clearance of dendrimers may limit theirapplications in drug delivery if not solved.

Micelles are composed of amphiphilic molecules that assemblespontaneously in water to form a lipid layer with a hydrophobic corethat can entrap poorly soluble drugs and a hydrophilic tail that is inequilibrium with the aqueous surrounding. Micelles are relatively smallwith a diameter range of about: 10 to 100 nm, 20 to 90 nm, 30-80 nm,40-70 nm, or 50-60 nm. that helps in deep tumor penetration but canstill escape renal excretion. They are considered biocompatible meaningthey are non-toxic to the human body. Micelles can be engineered towardsmore efficient drug targeting and prolonged blood circulation times byconjugating different ligands to their surface. Additionally, they canenhance drug distribution and pharmacokinetics by increasing drugstability and solubility, and decreasing its cytotoxicity to healthytissues. Polymeric micelles are highly used in drug deliveryapplications especially Pluronic copolymers due to their higher drugloading capacities, ability to overcome multi-drug resistance (MDR),lower CMC (enhanced stability) compared to other micelles, and theability to release drugs in response to specific triggers.

Regular micelles may only encapsulate hydrophobic drugs, even thoughrecently-reported core-inversible micelles may also encapsulatehydrophilic drugs. Another downside includes the recognition andelimination by the immune system, which can be neutralized using PEOcopolymer in the micelles structure, but this was found to interfere andprevent their interaction with cells, a process that may be necessaryfor a successful drug delivery carrier/system. Stability problemsemerged with incorporation into the bloodstream, where micelles arediluted beyond their CMC leading to unwanted early release of thetherapeutic agent. They might be stable at higher CMC values, but maynot be tailored to the body. Several other successful approaches havebeen suggested to deal with this drawback.

Liposomes as Nanocarriers

Liposomes can be used to entrap various types of molecules including:drugs, vaccines, plasmid DNA, peptides, hormones, antisenseoligonucleotides or ribozymes, nutraceuticals and cosmetics. Liposomesare one of the most widely used nanocarriers in drug delivery. Liposomesare spherically shaped phospholipid bilayers (lamellae), and inembodiments may have a diameter range of between about: 20 to 1000 nm,100-900 nm, 200-800 nm, 300-700 nm, or 400-600 nm in which each of thesemonolayers is constitutes of amphipathic molecules; that is ahydrophilic (polar) head and hydrophobic (nonpolar) tail, and can alsocontain other molecules such as cholesterol, carbohydrates and proteins.By shielding their hydrophobic domain, amphipathic lipids may formenclosed membranes in aqueous environments as shown in FIG. 3A, which isdescribed further in detail herein. The hydrophilic heads face theoutside of the double layer, whereas the hydrophobic tails come togetherto form the hydrophobic region, in which poorly water-solubleanti-neoplastic drugs can be loaded. The aqueous compartment inside thecore has the ability to entrap hydrophilic drugs. In embodiments, someof the chemotherapeutic drugs that can be loaded into liposomes includedoxorubicin, annamycin, daunorubicin, vincristine, cisplatinderivatives, paclitaxel 5-fluorouracil derivatives, camptothecinderivatives, and retinoids. At least some liposomal membranes areconsidered similar to some cells membranes structures, rendering themsafe to use in clinical trials (biocompatible and biodegradable). Insome instances, the properties of liposomes can be tailored to performvarious functions by controlling, for example, their lipid composition,particle size, surface charge, lipid membrane fluidity, and stericstabilization.

The two main types of phospholipids of liposomes are sphingomyelin andphosphoglycerides, in which they have sphingosine and glycerol as abackbone respectively. FIG. 1 illustrates a schematic drawing of theamphipathic molecule composition of phosphoglycerides and a lipidbilayer constructed from phosphoglycerides. Phosphoglycerides are themost common type of phospholipids forming liposomes found in nature;they consist of a phosphatidic acid 101 that is attached to ahydrophilic head group 102. Phosphatidic acid can include a glycerolbackbone in which two fatty acid chains 104, each has 14 to 24 carbonatoms in length, are attached to carbon-1 and carbon-2 of glycerol 103.And carbon-3 of glycerol 103 is esterified to the phosphate group 105 ascan be seen in FIG. 1, to form a head group. The head group isesterified to the phosphate group, and the head group may be, forexample: choline, serine, inositol, or as ethanolamine molecule leadingto different Glycerophospholipids: phosphatidylcholine (PC),phosphatidylserine (PS), phosphatidylinositol (PI), orphosphatidylethanolamine (PE), respectively.

Liposomes are classified, depending on their number of bilayers intomultilamellar vesicles (MLVs) and unilamellar vesicles. FIG. 2illustrates classification of liposomes based on size. In multilamellarvesicles (MLVs) more than one fluid compartment are present andseparated by lipid bilayers. As shown in FIG. 2, they range in sizebetween 500 and 5000 nm, while Unilamellar vesicles (ULVs) contain onlyone internal aqueous compartment. The later can be further classifiedinto small unilamellar vesicles (SUVs) that range in size between 50 and100 nm and large unilamellar vesicles with a size in the range of 100 to250 nm, respectively. Most liposomes traditionally used in drug deliverybelong to the SUVs type due to their higher capacity for drugs. Eventhough MLVs can exhibit slower drug release and higher loadingcapacities for hydrophobic compounds, they may have limited industrialapplications due to their heterogeneity in size, large diameter,multiple internal compartments, and inconsistent methods of preparation.

Liposomes can be also classified based on the stimuli that they areresponsive towards. Echogenic liposomes are rendered sensitive toultrasound by entrapping gas in their core, thus releasing theircontents as soon as ultrasound is applied. eLiposomes may containnanoemulsions that change phase from liquid to gas in response toultrasound, thus leading to subsequent drug release. Temperaturesensitive liposomes (TSL) may be triggered by hyperthermia. Liposomescan also be made sensitive to pH, in which they usually respond toacidic conditions, which is a characteristic of the tumor region, andlate endosome within the cell after liposomes endocytosis(internalization). In addition, it is possible to create light-sensitiveand microwave sensitive liposomes as well.

Nanocarriers may be administered to the body of a patient in variousways, including but not limited to: oral administration, intravenousinjection, intramuscular injection, or subcutaneous injection.Nanocarriers face many unexpected hurdles upon interaction with bodyfluids. They are naturally attracted to the liver by the action ofclearance. Liposomes can be engineered by covalently attaching moietiesto their surface in order to render them more stable, less immunogenic,better target the infected cells, increase blood circulation time,and/or protect them from degradation in the plasma. Liposomes bloodcirculation time depends on their size, their surface charge, and degreeof unsaturation of the lipid chains. Commonly used targeting moietiesattached to the liposomal surface include antibodies, hormones, andproteins.

FIGS. 3A-3D illustrate structures and evolution of liposomes. FIG. 3Aillustrates a hydrophobic drug 302 which may be trapped at thehydrophobic region of the liposome, and a hydrophobic drug 301 which maybe loaded in the aqueous compartment of the liposome. FIG. 3Billustrates liposomes after the attachment of antibodies 303 covalentlyto the surface groups and/or antibodies 304 hydrophobically anchoredinto the membrane. FIG. 3C illustrates stealth liposomes, where aprotective polymer 305 hinders opsonin proteins 306. FIG. 3D illustratesliposomes which includes both antibodies and a protective polymer, whereantibodies 107 are directly attached to the surface and antibodies 108are attached to the distal ends of the protective polymer.

In embodiments, sterically-stabilized liposomes (also referred to asstealth liposomes) may have the hydrophilic polyethyleneglycol (PEG)attached to their surface as shown in FIG. 3C. This protective polymermay help in avoiding hydrophobic interactions with plasmatic proteins(i.e. opsonin proteins 306 of FIG. 3C) and their subsequent adsorptionby the liposome membrane. This can reduce the trapping of liposomes bymacrophages of the mononuclear phagocyte system (MPS, also known as thereticuloendothelial system (RES)), in a process called opsonization,thus resulting in prolonged blood circulation time. Basically,increasing the hydrophilicity of the liposomes creates a steric barrierto prevent detection by the MPS and subsequently prevents liposomeclearance.

In some embodiments, stealth liposomes also have the enhanced ability tocross biological barriers and are used in the treatment of solid tumors.In certain embodiments, oating liposomes with PEG was found to increasetheir half-lives up to 12 to 30 hours in animal models and 21 to 54 inhumans. But the PEG polymer can also prevent cells interactions. Also,it was reported that some stability issues rose with PEG stealthliposomes. Due to their hydrophobic nature, the PEG polymers can actagainst the hydrophilic property of the head group and causedestabilization. To counteract this effect, a sufficient amount ofcholesterol (a rigidifying agent) can be added into stealth liposomes.Other coating polymers have been suggested but because of PEG's ease ofpreparation, relatively low cost, controllability of molecular weightand linkability to lipids by a variety of methods, it was widely used.It was reported that the size and the fluidity of the liposomes couldaffect its uptake by the RES, the smaller their size and the more rigidthey are, the better chance they have to avoid clearance. Anotherapproach to deal with liposomes clearance is to render them less foreignand more recognized as self-proteins by the macrophages of the MPS usingcoats of natural glycolipids, gangliosides. But it wasn't pursued due tosome difficulties.

As shown in FIG. 3B, in immunoliposomes, antibodies and their fragmentsmay be attached to the liposomes. Antibodies can identify antigensoverexpressed on the targeted cells surface. Each antibody binds to aspecific antigen, making it possible to selectively target that cell,hence avoiding undesired interactions with healthy cells. Most widelyused moieties for that purpose are immunoglobulins (Ig) and theirfragments, which can be attached to the liposomes by covalent bonds. Itshould be noted that they may still end up in the liver due toinsufficient time for the ligand on the liposome to interact with thetargeted receptor on the cell surface. This can be solved by increasingtheir blood circulation time, thus leading to the development ofsterically stabilized immunoliposomes. Sterically stabilizedimmunoliposomes can be synthesized by attaching the antibody directly tothe liposomes surface parallel to the PEG polymer, or successively tothe PEG polymer on the surface of the membrane, as can be seen in FIG.3D. Using the later methods will result in having both properties oflong circulation time and targeting ability.

Liposomes can also be modified with cell-penetrating peptides. Someviral proteins have the ability to penetrate cells through the proteintransduction phenomena, one such example is the TAT protein found inHIV-1, in which it mediates intracellular transport of nanoparticles.Using this method there is a better chance for nanoparticles to escapethe endosome and get into the cytoplasm. It has been proven using TAT asa ligand on liposomes surface facilitate its delivery into cells.

In certain embodiments, liposomes, as a drug carrier, are known to havefollowing advantages: 1) enhanced drug pharmacokinetics, distribution,and solubility, by preventing drug interaction with bodily fluids andearly degradation; 2) prolonged duration of drug exposure and controlthe drug release rate; 3) natural accumulation around the tumor areawhich increases the drug's concentration at the diseased site comparedto healthy tissue; 4) ability to be actively targeted to bind to cancercells more preferentially enhancing tumor uptake also intracellular drugdelivery; 5) possible modifications to make liposomes more appealing tobe used in many domains other than drug delivery, including diagnosis,regenerative medicine, and gene therapy; 6) the ability to control therelease of drugs, and to increase solubility of drugs; 7)biocompatibility and bio-degradablability and weak immunogenicity; 8)ability to overcome multi-drug resistance; 9) the ability to delivervarious types of drugs and increased loading capacity; 10) the abilityto be remotely triggered.

However, it is also known that certain currently available liposomeshave the following limitations as nanocarriers: enhanced accumulation atthe tumor site but less blood circulation time (more clearance);insufficient time to interact with the cancer cells due to ending up inliver and certain side interactions.

EPR Effect

The first generation of nanocarriers used in nanomedicine were based onthe EPR effect, also called passive targeting. EPR is defined as theenhanced vasculature and system permeability to molecules andnanoparticles in and around the tumor area; due to the defectiveness ofthe tumor vascular structure, and entrapping those particles forprolonged periods of time before their subsequent clearance by thelymphatic system; due to the impaired lymphatic system at the tumorsite. EPR may be applied to biocompatible lipidic particles andmacromolecules with large molecular weights, whereas low molecularsubstances are observed to return to the circulatory system bydiffusion. Usually, long circulation times (around 6 hours) are neededfor the accumulation of any drug due to EPR effects.

Enhanced tumor permeability is a physical phenomenon that depends onblood vessels morphological differences between normal and healthytissue; due to rapid angiogenesis. Normal blood vessels are linear andstacked regularly. But blood vessels in tumors have openings in theendothelium and are weak due to the lack of an external muscle layer,leading to high blood pressure in the tumor site. Tumor blood vesselsalso show polymeric leakage at the capillary level. These structuraldeviations and vascular permeability render cancer vessels leaky, and asa consequence, macromolecules and lipid particles are allowed toextravasate from the blood vessels into the tumor interstitial space andaccumulate as time passes by. Permeability is also enhanced by theincreased production of permeability mediators such as bradykinin,nitric oxide, prostaglandins, and VEGF (or vascular permeabilityfactor), in or near the most solid tumors. It is also known that theeffectiveness of EPR depends on the molecular size and/or weight. It wasalso reported that endothelium openings range from 300-4700 nm, in whichthey are not found in normal tissue. Generally, liposomes that rangefrom 90-200 nm are known to exhibit increased accumulation due to EPReffects.

Known measures suggested to enhance the EPR effect include raising thesystemic blood pressure, increasing NO concentration utilizingNO-releasing agents, and increasing kinin (bradykinin) concentrationusing ACE inhibitors. Further, in order for the penetrated materials toaccumulate, they need to escape clearance. This happens as a result offunctional defectiveness of the lymphatic system that usually removesforeign particles from the interstitial space.

Active Targeting

As passive targeting provided an approach to cancer treatment, itdoesn't exclusively target tumors, as inflammation also exhibits EPReffects. Additionally it does not guarantee intracellular uptake of thedrug. Moreover, the issue of molecules diffusing back to the bloodstreamis considered another drawback, and it has become clear that relying onEPR effects alone for targeting is not adequate. As have been mentionedelsewhere in the specification, cancer cells over-express some receptorson their surface vastly more than those of normal ones. This isgenerally due to cancer characteristics mentioned earlier, such asaccelerated proliferation, abnormal angiogenesis and some othermutations.

Active targeting in liposomes may include utilizing ligand-receptorlinks to increase cellular uptake of the nanocarriers throughreceptor-mediated endocytosis, where after EPR effects lead them closeenough to the affinity of the tumor tissue, the conjugated ligand on thesurface of the liposome is recognized by its receptor overexpressed onthe cancer cell surface, leading to increased efficient targeting.

Generally, most cancers over-express VEGF receptors, integrins, andvascular cell adhesion molecules which are all related to cancerangiogenesis. Folate receptors were also known to be extensivelyover-expressed in many tumors such as lung, brain, breast, kidney, andovary cancers, in order to improve their growth. Other cell divisionreceptors are human epidermal receptors (HER) family including EGFreceptors (HER1) that are commonly found in multiple cancer types suchas head and neck, bladder, ovarian, cervical, and oesophageal cancer.Also, the transferrin receptors are of importance in cell division, andare generally over-expressed in multiple tumors including braincapillary endothelial cells. Also, some cancers overexpress receptorsspecific to one type of the tumor cells, and these depend on themalignancy, location, and stage of the tumor.

Ligands for Targeted Drug Delivery

In certain embodiments, ligands used in ligand-targeted drug deliverymay include glycoconjugate, oligopeptide, nucleic acids, aptamers,carbohydrates, vitamins, whole proteins, peptides, and antibodies andantibody fragments against tumor markers.

The folate receptor can internalize its ligand into the cytosol. Uponattachment of the ligand, both the ligand and the receptor areinternalized and then moved to the acidic portion of the endosome (lateendosome) where de-attachment normally happens. The receptor is recycledback to the membrane, and the nanocarriers can release their contentsinto the cytosol. Folate group can be covalently attached to thephospholipid or cholesterol of the liposomal drug carrier, and it hasshown to reduce cardio-toxicity more than the free drug, inducecytotoxicity to targeted cells, and inhibits tumor development. It wasreported that attachment of folic acid to micelles, such as liposomes,(in the PEG copolymer side) entrapping a drug significantly increasedcellular uptake of the drug than those which are folate unconjugatedmicelles against KB cells over-expressing folate receptors.

It is reported that coupling PEG end of drug-loaded liposomes totransferrin significantly increased the cellular uptake by the cell linein vitro. It was also observed that OX26 monoclonal antibody (mAb) couldbe used to better target liposomes towards the transferrin receptor whenattached to the distal end of the PEG molecules conjugated to thesurface and coating the liposomes.

In certain embodiments, EGFR may gave various ligands; they include EGF,anti-bodies, antibodies fragments, aptamers, and EGFR-specificlow-molecular-weight peptides. FDA-approved antibodies with affinity toEGFR include Trastuzumab and the chimeric monoclonal antibody cetuximab.Several nanoparticles including liposomes have been modified usingcetuximab. Antibody fragments of cetuximab have also been used asmoieties for liposomes, due to their small size. The native ligand EGFhas a very high affinity for EGFR and showed strong cytotoxicity totumors, but could also lead to triggering signaling pathways of thereceptor and contribute to tumor development. Peptides used for EGFRtargeting include D4 and GE1. Anti-EGFR aptamers, a class of functionaloligonucleotides similar to the antibodies in their binding affinity,have been successfully used to specifically deliver gold nanoparticlesto EGFR.

Increased overexpression of vasoactive intestinal peptide receptors(VIP) on breast cancer cells make them successful targets for theVIP-conjugated nanocarriers. An increased intracellular uptake andsubsequent cytotoxicity have been reported upon using VIP as an activetargeting moiety in stabilized micelles.

Some integrins may be over-expressed in actively proliferatingendothelial cells. They have several ligands of extracellular matrix(ECM) proteins including fibrinogen, vitronectin, collagen andfibronectin. Therefore it has been suggested to target integrins of thetumor tissue using short peptides containing an arginine-glycineaspartic(RGD) site that mimics fibronectin and has high affinity towards theseintegrins. The RGD cell adhesion sequence is the cell attachment site ofmore than 20 integrins including integrin α_(IIb)β₃ and αvβ₃-integrins.Other peptides that bind to integrins represent slight variations of theRGD sequence, and may include but not limited to: the KGD sequence thatbinds specifically to α_(IIb)β₃ integrin, RHD sequence, PlateletEndothelial Cell Adhesion Molecule (PECAM, the endothelial cell marker)that binds to αvβ₃-integrins, or the NGR sequence that can bind tovarious RGD-directed integrins but has lower affinity than the RGDsequence.

Vascular cell adhesion molecules provide very highly specific targetreceptors due to that they are almost exclusively expressed on cancercells. These also bind to ECM proteins that are considered crucial formetastasis. Ligands used may include anti-VCAM-1 (vascular cell adhesionmolecule 1) monoclonal antibody.

Liposomes that have monoclonal antibodies or antibody fragmentsconjugated to their surface are called immunoliposomes, and theantibodies have high affinity towards specific antigens expressed oncell surfaces. Antibodies may bind to their corresponding epitope siteon the antigen over-expressed on many cancer cells. Some tumors arehighly immunogenic, and some are not, and the presence and the type ofthe antigen vary from one type of malignancy to another, whicheventually affects the efficacy of the immunoliposomal therapy. Asdescribed elsewhere in the specification, potential highly investigatedtargets for antibodies may include VEGF, EGFR, HER2, transferrinreceptors, and prostate-specific membrane antigen (PSMA).

HER2 receptors have an ability to internalize their ligands resulting inthe endocytosis of the antibody mediated nanoparticles. Trastuzumab isknown to be humanized monoclonal antibody for HER2. Nanoparticlescoupled with trastuzumab have been investigated for HER2 positive breastcancer. Another known chimeric monoclonal antibody is cetuximab whichhas a high affinity towards the EGF receptor (EGFR). Cetuximab-targetedgold nanoparticles were investigated for delivery of chemotherapeuticsto many cancers including pancreatic and colorectal carcinoma, andresults showed significant tumor growth inhibition. Cetuximabimmunomicelles were suggested as delivering vehicles for doxorubicinagent, as well as immunoliposomes conjugated with cetuximab to deliverboron in glioma cells overexpressing EGF receptors. Anti-transferrinreceptor antibodies include OX26 and R17217 monoclonal antibodies. Also,antibodies for Prostate-specific membrane antigen (PSMA) include J591monoclonal antibodies. Bevacizimab (Avastin) was successfullyimplemented in combination with chemotherapy in the treatment ofmetastatic breast cancer, in which it was targeted against VEGFoverexpressed as a result of angiogenesis. Lastly, rituximab, ananti-CD20 monoclonal antibody is used as a conjugate in nanoparticles totarget lymphoma tumors overexpressing CD20 receptors. Anti CD20receptors mAb are not considered internalized mAb in contrast to antiCD19 receptors mAb.

Due to the large size of monoclonal antibodies which could pose anobstacle for intracellular drug delivery, antibody fragments weresuggested because of their small size and similar affinity to theircorresponding antigens as whole antibodies do. Antibody fragments usedin nanomedicine include single-chain variable fragments (scFV) andantigen-binding fragments (Fab). Nanocarriers decorated with antibodyfragments exhibit reduced clearance by the RES and their small sizeallow for better penetration. scFV that bind specifically to an isoformof fibronectin was found to enhance the targeting ability of liposomes.Also, scFV-CM6 was found to bind specifically to a protein extensivelyoverexpressed on surfaces of tumor cells (TEM1) and was usedsuccessfully in making immunoliposomes. It was also reported thatefficient internalization was shown when liposomes conjugated withsingle-chain anti-EGFR antibody were used. Additionally, someantigen-binding fragments (Fabs) of monoclonal antibodies weresuccessfully used as conjugates to liposomes. These include Fabstargeting β₁ integrins that are overexpressed in lung cancer and Fab ofthe mAb anti-GD(2) that targets disialoganglioside which isoverexpressed on the surface of neuroblastoma cells. Also, human B-celllymphoma was targeted using immunoliposomes conjugated with mAbanti-CD19 or its Fab fragments. Finally, Fab fragments of trastuzumabwere used to target HER2 overexpressing breast cancer cell lines.Results in vitro showed increased cytotoxicity, and in vivo showedenhanced tumor growth inhibition.

Monoclonal Antibodies and Immunogenicity of Tumors

An antigen is any molecule that can interact with an antibody, and itsbinding site called epitope. An antigen can be a peptide, a lipid, acarbohydrate, a nucleic acid, or any other molecule. Any substance thatcan induce an immune system response is defined as immunogenic, allimmunogens are antigens, but not all antigens are immunogens (they alsoinclude allergens and tolerogens). Depending on the immunogen size,chemical, composition, conformation, and its “foreignness” they have theability to provoke an immune response.

Some antigens that are marked “self” do not stimulate an immuneresponse; these are normally expressed on normal healthy cells. Butmutations in cancer cells result in either altering these proteinsmaking them more foreign, or over-expressing them, in which both shouldlead to an immune response. Antibodies are naturally produced by B-cellsas a response to the antigen representation by helper T-cells.Antibodies belong to proteins of the blood called immunoglubins. Theyare classified into five different classes; IgM, IgD, IgG, IgA, and IgE.Each class has a similar component in their structure, and a smallvariable fragment (Fv) part (N-terminal (amino-terminal) domains) foundin the antigen binding fragment (Fab). That variable fragment is uniquefor each antigen-antibody binding. Antibodies consist of dual heavy andlight chains joined by disulfide bonds with average molecular weight of150 kDa.

Monoclonal antibodies are antibodies produced in a laboratory byculturing antibody-producing cells. Their production depends onimmunizing a mouse with a pathogen or any other immunogenic substance.These complex antigens may have many antigenic sites which result in theproduction of various antibodies in the blood stream for that onecomplex antigen. Each antibody is produced by a specificantibody-producing cell in the spleen. Antibody-producing spleen cellsmay be fused with immortal myeloma cells to have hybrid cells thatcontain both immortal properties and antibody secreting ability ofparent cells. These can then produce polyclonal antibodies. Eachhydridoma may be then cultured individually to produce separated clonesthat secrete one specific type of antibodies, called monoclonalantibodies. Using monoclonal antibodies, one can ensure the precisebinding to only one antigenic site of the tumor, without concern aboutwhether or not it will affect other targets.

Monoclonal antibodies can be used as homing devices to guidenanocarriers to tumor targets (receptors) and hook with them, or inimmunotherapy to interfere with cell signals and specific moleculesfunctions that are necessary for tumor growth and angiogenesis aspreviously discussed in targeted therapy.

Limitations to monoclonal antibodies include expensive production,immunogenicity, and limited conjugation density on nano-carriers due totheir large size. Also, mouse-derived antibodies were shown to inducesome allergic-like reactions when used in humans, which raised the needfor creating chimeric or humanizing murine-derived antibodies, or aimingfor producing fully human monoclonal antibodies. Chimeric monoclonalantibodies are considered less compatible with humans than humanizedones; they have the variable fragment from a murine source and theconstant region from a human. While humanized monoclonal antibodies haveonly the complementary determining regions of the variable regions(CDRs) from a murine source. Fully human monoclonal antibodies weredeveloped using phage-display technologies. Usually, fragments ofantibodies display less immunogenicity.

Modifications to mAbs may be needed for conjugation purposes. Sites forchemical binding in antibodies, and proteins, in general, include thiolgroups (sulfhydryl groups) that are found in cysteine residue of theprotein, amine groups that are located in the lysine residue, andcarbohydrates. Usually, sulfhydryl bonds in proteins are found in theirreduced version as disulfide bonds (in cystine), which first need to beactivated into a free thiol group in order for the conjugation to besuccessful. These modifications are known to affect the antigen-antibodybinding sites except for the carbohydrate modification. For disulfidemodification at low pH, damage control can be achieved.

Generic names of monoclonal antibodies used in therapy are based ontheir targets and type of monoclonal antibody used. One of skill in theart will recognize that a “ximab”, “zumab”, or “mumab” suffix indicateseither chimeric human-mouse antibodies, humanized mouse antibodies, orfully human antibodies respectively. A stem in the middle indicatingtheir type of target is a “ci(r)”, “t(u)”, “li(m)”, “tin”, or “zom” toindicate a circulatory system target, a tumor target, an immune systemtarget, a tyrosine kinase inhibitor, or a proteasome inhibitorrespectively.

Trastuzumab, also called Herceptin, is a humanized IgG(1) kappamonoclonal antibody (145.5 kDa) with high affinity towards the HER2receptor overexpressed in breast tumor cells. Trastuzumab can preventHER2 hetero-dimerization and stop cells signaling related to tumordevelopment. Thus it has been used as a treatment in immunotherapy asdescribed elsewhere in the specification. Trastuzumab has been shown toreduce the risk of recurrence when used as adjuvant therapy, and alsoaugment the effects of chemotherapy. Trastuzumab was commonly used incombination with Paclitaxel, Docetaxel, Navelbine, Gemcitabine, andCapecitabine. Pertuzumab is another monoclonal antibody specific to HER2but binds to a different epitope than trastuzumab.

Ultrasound as a Trigger for Drug Release

Triggering mechanisms may allow for controlling the release at tumorsites, resulting in dismissing side effects on healthy cells, avoidinginducing drug resistance that is due to long accumulation time. They mayalso facilitate penetration into the tumor, and endosomal release.Releasing the drug too early or too fast may result in damaging healthycells, while releasing the drug too slow or too late won't allow for theconcentration to reach the cytotoxic dose, thus controlled release maybe needed. Once the drug nanocarriers reach the tumor site, spatial andtemporal controlled release may be obtained by ultrasound, such amechanism is widely used for triggered release due to its low cost,safety, and focused feature.

Ultrasound is a cyclic sinusoidal acoustic wave that has high-pressurephases (compression) at the upper peaks and low-pressure phases(refraction) at the lower peaks. It propagates through the medium, bytransferring of energy through the oscillation of particles, thus itpropagates faster in solids than in fluids. The ultrasound frequencyranges are above the human hearing range (20 kHz). Attenuation is theloss of intensity as the wave travels through some medium, where energyis lost either by absorption or transferred to other forms of energy.

Parameters of ultrasound that are of importance in triggered drugrelease include its frequency, intensity (power density), and mode ofoperation. Low-frequency ultrasound (LFUS) which is generally less than1 MHz is applied to trigger release. High-frequency ultrasound (>5 MHz)has been used in diagnostic imaging in medicine for ages. Generally, asthe frequency of the applied ultrasound increases, less penetration intotissue occurs. Further, at low frequencies cavitation increases, asdescribed further elsewhere.

Ultrasound intensity is the energy carried per cross-sectional area ofthe applied beam. Low-intensity ultrasound usually doesn't inducehyperthermia in contrast to high-intensity ultrasound (HIUS) that isfrequently used as a treatment of cancer as previously described, wherehigh temperature has the ability to damage cells in the site where theyare targeted towards. Several studies report a proportional relationshipbetween drug release and US power intensity. The mode of continuouslyapplying ultrasound may be used in triggered therapy as well as thediscontinuous mode (pulsed mode), where ultrasound has on and offperiods for specific time spans.

Ultrasound has thermal effects and non-thermal effects (mechanicaleffects). Thermal effects (hyperthermia) previously described are theresult of energy dissipation of HIUS into thermal heat rising tissuetemperature. Mechanical effects result from the acoustic wavepropagation and pressure variations. One such effect is acousticcavitation, which is the formation of gas bubbles in a liquid, due tochanges in pressure. Cavitation depends on the intensity of theultrasound, and it only occurs at a certain threshold. At low-pressureamplitude, the gas bubbles exhibit stable oscillation, in which theycontract and expand slightly, this is referred to as stable cavitation.On the other hand, inertial cavitation results from high-pressureamplitude that leads to gas bubble collapse. The bubbles increaserapidly in size until they reach their resonant size, at which then theycollapse resulting in: high pressure and temperature, sonic jet of fluid(near solid surfaces) is produced and damages nearby cells, shock wavesthat shear open the cells, and the formation of new small bubbles thatreinitiate the cycle. Stable and inertial cavitation can occur in thesame situation following each other, they are not separate phenomena.Another mechanical effect is acoustic streaming which is a direct resultof the US wave propagation through some medium. In acoustic streaming,particles move in the direction of the flow, resulting inmicro-streaming, bulk-streaming or both. The latter is considered apowerful mechanism that facilitates the delivery of drugs.

One of important acoustic parameters is the mechanical index (MI), whichis the probability of collapse cavitation to happen. In triggered drugdelivery, the aim is to find the optimum ultrasound parameters thatpermit enhanced drug delivery without harming healthy cells. This couldbe better achieved if we understood the mechanism by which enhancedtriggered delivery works. Several mechanisms could be the cause of thetriggered drug delivery: disruption of the drug nanocarriers;enhancement of drug distribution in tumor tissue; enhanced intracellulardrug uptake by endocytosis; and/or increase in cellular uptake of thenanocarriers by enhancing the cell permeability.

The first possible mechanism could be that shear stress resulting fromboth wave pressures and cavitation can lead to disruption of thenanocarriers membrane. Ruptures resulting from cavitation areparticularly important for site triggered drug delivery, to avoid drugrelease near healthy cells. In the second mechanism, microstreaming mayenhance the distribution of the encapsulated drug by diffusion throughthe tumor tissue. Moreover, cavitation has an enhancement effect on themotion of the fluid near the tumor cells, in which drug dispersionoccurs. Gas bubbles of the cavitation phenomena can pull densermaterials (nanocarriers for example) towards them, resulting in theirrupture. In the third mechanism, the uptake of micelles into tumor cellswas reported in several studies, suggesting nonspecific endocytosis.Additionally, cell membrane permeability may be a direct result ofevents resulting from cavitation. Shock waves, sonic jets, andmicro-streaming may induce pore formation on cells membranes and thusfacilitating the drug uptake into cells.

Modeling the Release Kinetics

The concept of controlled release lies within the fact that an initialdose of the drug is needed and then a further slow release, to maintainthe drug therapeutic level as long as possible. Control release for adrug delivery system is needed since some drugs release too fast fromcarriers, before getting to the tumor, and others too slow that thetherapeutic effect can't take place.

Controlled release aims to reduce the frequency of the treatment andincrease patients comfort level. For this purpose, modeling of therelease kinetics is needed in the optimizing stage, where the patternsof the release can be predicted without the need for unnecessary studiesor experiments. It may also provide some insight into the mechanisms bywhich the drug is released and some other physical aspects as well.Several release mechanisms are common, they include but not limited to,release by drug diffusion through the polymer membrane, by thedegradation of the polymer, or by chemical disassociation of the drug.

For example, the kinetics of drug release of liposomes inPhosphate-buffered saline can give an idea about the release behavior invivo, thus reducing studies done in vivo. Models describing drugdissolution differ based on their assumptions, but they can becategories as follows: slow zero order, first order, and ones that startrapidly and then reduce to either of the previously mentioned types.Kinetics can be influenced by the type of drug, particle size,solubility, and the amount used. Nine models will be used in this thesisto discuss the release kinetics of the model drug calcein under lowfrequency ultrasound.

Breast Cancer and Immunoliposomes Targeted Towards HER2

In some embodiments, doxorubicin (“Dox”)-loaded immunoliposomes (ILs)may be provided. Sterically stabilized liposomes (70-100 nm in diameter)may be conjugated to anti-HER2 mAb fragments. Delivery of theseimmunoliposomes into HER2 overexpressing cells may result inintracellular uptake of 600 times higher than nontargeted stealthliposomes, but they may exhibit similar uptake in non HER2overexpressing cells, which may demonstrate the effectiveness oftargeting moiety. Also, ILs may exhibit 700 times more accumulation intargeted tissue than in negative cells. In vivo studies conducted onxenograft that overexpresses HER2, reported that ILs loaded withdoxorubicin yielded improved anti-tumor activity in contrast to allother treatment options used which included: free Dox, free mAb(trastuzumab), liposomal Dox, free Dox conjugated to the mAb, andliposomal Dox linked to trastuzumab.

However, tumor tissue levels of ILs and liposomes may be the same, butILs may exhibit intracellular uptake opposite to non-targeted liposomesthat accumulated in the tumor stroma, which may result in 10-30 timeshigher cytotoxicity. Also, administration of ILs may not increaseclearance, hence showing that anti-HER2 mAb fragments may not affect thestabilization or the non-immunogenicity of sterically stabilizedliposomes.

In one instance, immunoliposomes (140 nm in diameter) may be conjugatedwith trastuzumab mAb to deliver both Paclitaxel (PTX) and rapamycin(RAP) therapeutic drugs into 4T1 cells that are triple negative breastcancer cells and SKBR3 cells which are HER2 positive breast cancercells. The encapsulation efficiency can be about 56% and 70% for PTX andRAP respectively, and the conjugation of Trastuzumab can be above 70%using a thioether bond. In embodiments, cytotoxicity of SKBR3 cells forthe ILs can be increased compared to the control liposomes (non-targetedliposomes). This can be a result of the enhanced uptake mediated by themAb bond to the HER2 on the cells. The in vivo study investigated theimmunoliposomes co-loaded with both drugs, control liposomes, andsolution of PTX/RAP against human xenograft HER2 overexpressing tumors,and immunoliposomes showed better anti-tumor growth activity. RAP canincrease PTX induced apoptosis, hence produce synergetic effects in thepresence of trastuzumab.

Stability of actively targeted liposomes may not be affected much incirculating conditions. It has been reported that liposomes incirculating conditions leaked 20% of their contents after 5 hours and42% after 8 hours, while liposomes in cell culture conditions leaked 5%after 5 hours and 9% after 8 hours. Thus liposomes may be consideredstable in circulating conditions in times up to 8 hours.

Antibody Conjugation Methods

Attachment methods of targeting ligands to liposomes may includecovalent and/or noncovalent bonds. The attachment done to the distalends of the PEG-PE anchor may be more efficient than linking directly tothe surface of the liposome. The approach of conjugating the ligand toliposomes after their synthesis may be better than linking them tolipids prior to liposomes synthesis.

In certain embodiments, types of linkages used in the conjugationmethods may include, for example, thioether bonds, disulfide bond, amidebonds, Hydrazide bonds, and crosslinking primary amines. It has beenreported that ILs-PEG-mAb linkage displayed increased binding, butreduced internalization compared to ILs-mAb linkage (but still containsPEG on their surface parallel to the mAbs). It has been also reportedthat attaching the mAbs to distal ends (ILs-PEG-mAb) showed that bindingwas independent of the PEG density. Additionally, increasing mAb densityon immunoliposomes has been reported to enhance binding andinternalization. A conjugation efficiency of 70-90% has been reportedfor using a thioether covalent bond in conjugation.

Also, stealth immunoliposomes may be prepared with trastuzumab Fabconjugated to the surface for one approach and conjugated to the distalends of PEG chain for another approach, both in which they use thioetherbonds. Increasing PEG density was reported to decrease the binding withthe first approach but did not affect the second approach. Also, it wasreported that binding and internalization was much higher in HER2positive cells than in negative ones for both approaches.

In some embodiments, a thioether bond in which the liposomes wherethiolated instead of the antibodies, may be used. When the attachmentwas done directly to the stealth liposomes surface, the PEG polymer mayaffect the attachment efficiency by hindering the antibodies away fromthe surface. So the attachment may be made on the distal ends of thepolymer, and to get high conjugation efficiencies. A summary ispresented in Table 2 that shows an embodiment of conjugationefficiencies of thioether bonding strategies with and without PEGpolymer on 100 nm liposomes.

TABLE 2 Antibodies conjugation efficiency using two different strategiesof applying the thioether bond, with changing the binding site and mPEGpolymer % on liposomes surface Conventional method Andibodies are whereAntibodies are maleimided and thiolated and liposomes or PEGSetup/strategy liposomes maleimided polymers are thiolated At thesurface with 63% 10% 5% mPEG At the surface with 72% 69% 0% mPEG At theend of the — 61% PEG with 4% mPEG on the surface At the end of the — 60%PEG with 0% mPEG on the surface

Thioether bonds may require pre-derivatization of both liposomes andantibodies, which is considerably complicated and not feasible when theantibody is especially expensive. A “Bendas protocol” may be used, wherea cyanuric chloride acts as a linkage between the PEG distal end and theantibody to prepare immunoliposomes. This method required no priorderivatization to the antibody and no extra chemicals as well. Bindingefficiency for the immunoliposomes was also established which means noharm was done to the binding site. Immunoliposomes stability was alsoconfirmed. Usage of cyanuric linkage to form immunoliposomes in acontinuous process has been also reported.

Calcein for Ultrasound Triggered Drug Delivery

Ultrasound release may be studied under the effect of frequency andpower density when the liposomes nature is not changing. The liposomesparameters affecting ultrasound include, for example, lipid ratio,surface charge, and PEG polymer density.

Calcein (a model drug) release from liposomes has been reported to behigher at LFUS than at HFUS, and the amount released was reported toincrease with increasing exposure time and the mechanical index,possibly due to mechanical effects rather than thermal effects.

The dependency of the release on the liposome membrane structure wasalso reported in dox-loaded liposomes under LFUS effects; 30% higherrelease using DOPE based liposomes than DSPE based liposomes.Accordingly, DOPE-based liposomes may be sonosensitive lipids.

It was also reported that PEGylated liposomes showed 10 fold morepermealization upon exposure to LFUS than the control non-PEGylatedliposomes, due to the absorption of energy by the PEG groups which areconsidered sonosensitive.

It was reported that estrone-targeted calcein-loaded immunoliposomesupon exposure to LFUS may exhibited higher initial release rates thanthe non-targeted ones, but the same final release rate for bothliposomes types.

Method for Synthesis of Immunoliposomes

Sterically stabilized liposomes with functional groups at their ends maybe synthesized, using a mixture of lipids. In certain embodiments, thefunctional group may be related to the type of linkage wanted and it maybe placed at the end of the PEG chain on the liposomal surface, hencethe ligand will be attached to the distal end of the PEG chain after theformation of liposomes. Lipids may be first dissolved in an organicsolvent, then dried until a lipid film forms. The drying condition candepend, for example, on the volume of the sample and the solvent used.After that, the lipid film may be hydrated with a suitable material(e.g., distilled water or the encapsulating material), and this may leadto the formation of multilamellar vesicles (MLVs) liposomes. In order toconvert it to unilamellar vesicles (ULVs), several techniques forapplying mechanical stresses may be used including sonication orextrusion. Subsequently, the liposomes may be purified to get rid ofunreacted substances and formed micelles, for example, by gelchromatography, based on size (micelles and other molecules are verysmall compared to liposomes). This may yield the control liposomes. Forthe actively-targeted liposomes, one more step may be needed to attachthe antibody to the functional groups at the distal ends by a suitablereaction depending on the type of bond, which can be done, for example,with Bendas protocol described herein.

For simple, fast, and clean ligand binding, cyanuricchloride-PEG-liposomes may be used (cyanuric chloride being thefunctional group). This is because the resulting bond would not damagethe antigen binding site on the antibody, thus would not prevent theirspecific activity. The process may involve non-toxic materials and maynot require the pre-modification of the monoclonal antibody.

In certain embodiments, attaching the antibodies to the liposomesdirectly can prevent their activity and also increase the clearance ofliposomes by the reticuloendothelial system (RES), but conjugatingantibodies to the distal ends of PEG chains may yield long-circulating,fully functioning immunoliposomes. In some instances, it can alsoenhance the binding efficiency of the ligand to the liposome.

Materials for the Synthesis of Immunoliposomes

In some instances, dipalmitoylphosphatidyl choline (DPPC) and/or1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG(2000)-NH2) may be used as phospholipids for theimmunoliposomes, in order to prevent chemical degradation by oxidationwhich could lead to increasing the permeability of the bilayers.Oxidation can also be minimized using high-quality lipids and avoidinghigh temperatures. The choice of the buffer used, the pH, thetemperature, and the charge of the liposome may affect the hydrolysis ofthe phospholipids.

In some instances, the phospholipids may be selected to have suitablecholesterol molar ratio, which affects the stability of the liposomes.The addition of cholesterol to saturated lipids increases their fluidity(in contrast to unsaturated lipids), and thus gaps that are formed inthe lipid bilayers due to the trigger will reclose too soon. But theaddition of cholesterol may reduce the phase transition temperature ofthe lipid mixture.

The ratio of PEGyhlated lipid to non-PEGylated lipid may affectaggregation or accumulation of immunoliposomes, and in some instances,the ratio of PETyhlated lipid to non-PEGylated lipid may be below 6%. Insome instances, the ratio of PETyhlated lipid to non-PEGylated lipid maybe below about 10%, 5%, or 1%. The length of PEG chain for thePEGyhlated lipid may be selected such that the binding ofimmunoliposomes to the target may not be prevented or obstructed. Theprotein density of the liposome may affect affinity of the liposome toits target. In some instances, the protein density may approximately be7.5-30 molecules per liposome vesicle to yield a strong affinity towardsits target.

In certain embodiments, the gel-liquid crystalline phase transitiontemperature (T_(m)) of DPPC bilayer is 41° C., while it is 74° C. forDSPE, so the operating temperature for the preparation of liposomes maybe between 41° C. and 74° C. In some instances, the operatingtemperature may be between 45° C. and 70° C., between 50° C. and 70° C.In some instances, the operating temperature may be around 60° C. Thisis reasonable because the resulting lipid transition temperature rangesfrom 41 C.° for pure DPPC to 43 C.° for 15% DSPE-PEG (2000) lipidmixture, and the cholesterol helps to lower the transition temperatureas well.

In some embodiments, a fluorescence marker may be used as a model drug,instead of doxorubicin, for calcein release experiment, because thelatter is highly toxic and significantly more expensive. Also, calceinmay be generally used to model hydrophilic drugs, and it is easilydissolved in the lipid solution after adjusting the buffer pH. Some ofthe materials used in this work and their properties are listed in Table3.

TABLE 3 Properties of the reagents used in the synthesis MolecularTransition Material/properties weight (g/mole) temperature (° C.) DPPC734.039 41 DSPE 748.08 74 DSPE-PEG(2000)- 2790.486 — NH2 cholesterol386.65 — Trastuzumab 145531.5 —

Preparation of DSPE-PEG-NH₂ Control Liposomes

In some embodiments, control liposomes may be prepared usingcholesterol, DPPC, and DSPE-PEG(2000)-NH₂ at molar ratios of 30:65:5,respectively. The dipalmitoylphosphatidyl choline (DPPC) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG(2000)-NH₂) may be obtained from Avanti PolarLipids Inc. (Alabaster, Ala., USA). Cholesterol may be obtained fromAlfaAesar (Ward Hill, Mass., USA). The reagents may be dissolved in 4 mlchloroform to a final concentration of around 5-20 mg lipid/ml solventin a round-bottom flask at 60° C. Chloroform may be obtained fromPanreac Quimica S.A. (Spain). In some instances, the final concentrationmay be 10 mg/ml. Then chloroform may be dried in a rotary evaporatorunder vacuum for 15 minutes, until a thin film was observed on thewalls. After that, the lipid film may be hydrated using 2 ml of 30 mMcalcein solution and the pH may be adjusted to 7.4. Calcein disodiumsalt, and the bicinchoninic acid (BCA) kit may be obtained fromSigma-Aldrich Chemie GmbH (Munich, Germany). Then the solution may besonicated at 40-kHz using 100% power for 15 minutes in a sonicator bath(Elma D-78224, Melrose Park, Ill., USA).

The liposomal solution may be then extruded three times using 200-nmpolycarbonate filters (Avanti Polar Lipids, Inc., Alabaster, Ala., USA).Lastly, for the removal of free calcein and spontaneously formedmicelles, purification using size exclusion chromatography on a SephadexG-100 column was performed, after equilibrating it with borate buffer pH8.5. Sephadex G-100 may be obtained from Sigma-Aldrich (Sweden). All ofthe previous steps may be performed at 60° C., and/or above thetransition temperature of the lipid. Finally, dense liposome fractionsmay be collected and either used to prepare immunoliposomes, or storedat 4° C. after changing their buffer to PBS pH 7.4.

Preparation of Sterically Stabilized Immunoliposomes with Antibody-PEGLinkage

Preparation of sterically stabilized immunoliposomes with antibody-PEGmay be done in two steps, first modifying the liposomes with cyanuricchloride (C.C.), and then adding the antibody at a pH 8.5 to be linkedto the cyanuric chloride. Liposomes prepared as described above made beused here for the modification with cyanuric chloride. All the steps maybe conducted in an iced bath. First, cyanuric chloride may be dissolvedin acetone to make a 10 (mg/ml) solution. Then, 9.23 μl of that solutionmay be diluted in 0.5 ml di-ionized water, because alcohol may bedestructive to liposomes. The diluted solution may be added to 1 mlliposomes solution, to achieve 1:1 molar ratio of cyanuric chloride toDSPE-PEG-NH2 respectively. The reagents may be stirred at pH 8.5 and 0°C. for 3 hours to allow for the nucleophilic substitution of chlorideparticle on the cyanuric chloride with the proton on the NH2 group onliposomes. Then after that, trastuzumab may be added in excess amounts(e.g. 1 mg) after being dissolved in 0.5 ml borate buffer (pH 8.5). Thisreagents with trastuzumab may be kept stirring overnight to allow toreach completion, where the N-terminus on amino acids of the trastuzumabmay be linked to cyanuric chloride. Subsequently, the liposomes may bepurified to remove excess trastuzumab and any free calcein in aSephacryl S-200 HR column equilibrated with PBS (7.4 pH) and eluted withPBS (pH 7.4). The liposomes may be collected and stored at 4° C. untiluse.

Determination of Size of Liposomes

Size distribution of immunoliposomes may be determined in several ways,including, but not necessarily limited to, dynamic light scattering(DLS), electron microscopy, or right-angle light scattering andturbidity. For example, the mean size of liposomes may be determined atroom temperature by DLS using DynaPro® NanoStar™ model from WyattTechnology Corp. (Santa Barbara, Calif., USA). Viscosity andconcentration of liposomes may be measured as parameters, considering amedium viscosity of 1.020 and medium refractive index of 1.333.

First, the liposome samples may be diluted with PBS pH 7.4 and filteredusing 450-nm PVDF disk filters, then placed inside the machine foranalysis, where a laser is directed toward the sample. The machine maydetect fluctuations in the intensity of the light scattered, due tomovement of particles. This intensity may differ for large particlescompared to smaller ones, and based on a previous calibration process,measurements of size distribution can be obtained. Acceptable readingsmay correspond to <20% poly dispersion (PD) and the relatively low sumof squares (SOS), which may be all shown on the software used. Dataobtained from the software may be analyzed using two fits: cumulant andregularization. Cumulant fit analysis assumes the presence of one model,i.e. a uniform size distribution, however, this may be unlikely, sincepossible existence of micelles or other impurities. Therefore, theregularization fit may be used, and the particles may be assumed tobehave as multimodal particles distribution.

Zeta Potential Determination

Zeta potential determination measures the magnitude of the charge on theparticle surface. The measurement may be done using electrophorecticmobility of dispersion using a Zetasizer 3000 HSa equipment. Zetapotential with values between 25 mV and −25 mV indicate the tendency forparticle aggregation due to Van Der Waal inter-particle attractions.Values outside this range indicate the stability of the nanoparticle.The zeta potential may indicate the successful modification of thenanoparticles surface. Control liposomes may indicate negative valuesdue to the presence of the hydrophilic PEG polymers, but activelytargeted liposomes may counteract the charge of the PEG polymerresulting in an increase in the zeta potential (towards the positivedirection) if the binding process was successful.

Liposome Concentration Quantification

Liposome Concentration may be determined using a Stewart assay. InStewart assay, the detection of phospholipids may be based on complexformation between ammonium ferrothiocyanate and phospholipids, which maybe detectable in spectra at 485 nm. The complex is insoluble inchloroform, whereas the phospholipids are. Mixing ammoniumferrothiocyanate and phospholipids, and separating the phases may resultin a lower layer of chloroform in which the complex formed is dissolvedin, and an upper layer of the remaining. Detecting the spectraabsorption of the lower layer after separation may indicate thephospholipids concentration in the sample. The assay may be sensitive tosmall amounts down to 0.01 mg lipids in 2 ml chloroform (0.005-0.05mg/ml). Diluted samples can be used if needed and then the results canbe adjusted with the dilution factor.

A calibration curve for the DPPC may be prepared with increasingconcentrations from 0.0025 (mg/ml) to 0.025 (mg/ml). The liposomessamples may be dried in vacuum and then dissolved in chloroform, with adilution factor of 20. Then the solution may be sonicated to properlydissolve and break the liposomes to its constituent lipids. Sixreplicates may be made by adding specific amounts of lipid samples to 2ml ammonium ferrothiocyanate. Then the mixtures may be vortexed for 20seconds for proper mixing. And after that, the mixtures may becentrifuged to separate the chloroform layer. Subsequently, the lightabsorption at 485 nm may be read using a spectrofluorometer and resultsmay be used to calculate the DPPC concentration in each sample. Anaverage of the six measurements may be taken. Liposomes may be formedmainly by DPPC so the amount of the NH2-PEG-DSPE lipids may be safelyneglected.

Antibody Conjugation Confirmation Using BCA Assay

Protein conjugation efficiency may be determined in several ways,including, but not necessarily limited to the BCA assay (Smith assay),Lowry protein assay, Bradford protein assay (spectroscopic analyticalprocedure) or biuret test. BCA compared to Lawry assay may be simplerand allow for more flexibilities. And BCA may be more objective thanBradford assay since at higher temperature peptide bonds begin to takepart in the reactions. The BCA assay is based on two-step reactions. Thefirst is the reduction of copper Cu²⁺ to Cu¹⁺ upon interacting withamino acids and peptide bonds. The second is a change in color fromgreen to purple upon interacting with BCA reagent. This purple coloredcomplex may highly absorb light at 562 nm. The amount of reduced coppermay be proportional to the amount of protein in the sample.

In certain embodiments, using the micro BCA assay, to determine totalprotein concentration in a solution, working reagents A, B, and C may beadded together at molar ratios of 25:25:1, respectively. One milliliterof the resulting solution may be added to one milliliter of the buffer(e.g. PBS) and 100 μL of the sample (liposomes solution). Then, they maybe mixed for 30 seconds using the vortex machine, and subsequentlyincubated at 60° C. for 1 hour. A calibration curve to compare theabsorption spectra with protein solutions of known concentrations cangive direct concentration measurements.

Conjugation efficiency can be determined as the ratio of the amount ofprotein in immunoliposomes after purification to the ones beforepurification. Sometimes purification may not be 100% efficient, suchthat some free antibodies that were not attached can be detected in theassay. To overcome that, control liposomes may be prepared by simplymixing them with the antibody without performing the reaction, and thenthey can be purified. The difference in the protein amount between thetwo samples (control liposomes and immunoliposomes) may indicate thenumber of attached antibodies, not the free ones. Six replicates perliposome batch may be used to confirm the amount attached. The proceduremay be repeated for three batches of liposomes to confirm the attachmentand the consistency of the results.

Evaluation of Formulation Morphology by Transmission Electron Microscopy(TEM)

First, the sample may be placed onto a copper grid coated with a carbonmembrane. Then excess liquid may be removed after two minutes to allowfor adsorption. Then the sample may be dried at room temperature. Fornegative staining, a drop of 1% (w/v) aqueous solution of uranyl acetatemay be added. The surface morphology may be analyzed in a JEOL 2010 TEMwith 100 kv accelerating voltage.

Cytotoxicity

In determination of cytotoxicity of nanocarrier carried drugs, trypanblue may be used to distinguish between viable and dead cells. Viablecells membranes are very selective and do not allow the penetration ofthe Trypan blue dye inside the cells, in contrast to dead cells that do.To quantify cells uptake and determine the binding of immunoliposomes tocells, flow cytometry will be used. First, two types of cell lines(normal breast cells and HER2 overexpressing breast cancer cells) may becultured in 6-well plates. Then, they may be incubated with theliposomal solution at 37° C. for 1 hr. The wells will be trypsinized andcentrifuged and finally washed with PBS to remove floating unboundedliposomes, and analyzed in a flow cytometry.

Determination of the Number of Trastuzumab Molecules Attached to EachLiposome

The number of antibody molecules attached to each liposome may bedetermined assuming an average radius of liposomes of about 100 nm,which indicates that each liposome vesicle have around 80,000phospholipid molecules. With the knowledge of the molecular weight oftrastuzumab and DPPC, the number of trastuzumab molecules per vesiclecan be calculated after quantifying the protein and lipids amounts usingBCA assay and Stewart assay.

Release Experiments

Calcein release rate may be dependent on multiple factors, including butnot limited to liposomes composition, fluidity, permeability, andbending elasticity. Upon US triggered drug release, the release can beaffected by multiple factors, including but not limited to the mode ofoperation, the power intensity, the duration of the pulse, and liposomescomposition, gas encapsulated, and concentration in the sample. Thus,various power densities may be tested to achieve optimal drug release.As described elsewhere in the specification, it has been reported thatthe release at LFUS obtained better results than HFUS. For example,release at 20 kHz may be superior to 1 MHz and 3 MHz.

Continuous Release Experiments Using Phosphorescence/FluorescenceSpectrofluorometer

To trigger the release of the calcein, a model for hydrophilic drugs,from liposomes, 20-kHz LFUS may be used. The amount released can bequantified by fluorescence changes using QuantaMaster QM 30Phosphorescence Spectrofluorometer (Photon Technology International,Edison N.J., USA). Calcein is a fluorescence molecule and has anexcitation and emission wavelengths of about 495 and 515 nm,respectively. It may be used as an indicator for the liposomal leakageand drug release as follows: when it is encapsulated in a fluorescentquenching concentrations (≥30 mM), no fluorescent can be detected, butupon releasing to the aqueous surrounding solution due to acousticallytrigger release, it is diluted and the release can be measured bymonitoring the increase in fluorescent. Calcein fluorescence isdependent on pH at acidic conditions (pH<4.5), but independent at pHvalues ranging between 6.5 and 10. Release experiments may be conductedat a pH of 7.4.

The synthesized liposomes may be diluted using a solution of PBS at a pHof 7.4 in a fluorescence cuvette and placed inside thespectrofluorometer. For ultrasound exposure, 20-kHz ultrasonic probe(model VC130PB, Sonics & Materials Inc., Newtown, Conn.), may beinserted 2 mm in the cuvette, such that it does not cross the path ofthe emitted light through a special opening in the spectrofluorometer.Then, for data normalization, the initial fluorescence concentration(F_(o)) may be measured for 60 seconds before sonication. Then,ultrasound may be applied in a pulse mode, with 20 seconds “on” and 10seconds “off” periods, until fluorescence concentration plateaus.

To normalize release, 2% (w/v) Triton X-100 may be added to achieve afinal concentration of 0.48 mM. The surfactant may be used to lyse theliposomes and fluorescence concentration (F₁) to achieve 100% releasemay be monitored. These steps may be repeated for three different powersettings (20%, 25% and 30%), corresponding to three different powerdensities 7.46, 9.85, and 17.31 (mW/cm²). To calculate the dimensionlessfluorescence concentration at a given time, the following equation maybe used:

$\begin{matrix}{{{Cumulative}\mspace{14mu} {fraction}\mspace{14mu} {of}\mspace{14mu} {drug}\mspace{14mu} {released}\mspace{14mu} \left( {C\; F\; R} \right)} = \frac{F_{t} - F_{0}}{F_{1} - F_{0}}} & {{Eq}.\mspace{14mu} (1)}\end{matrix}$

The data for three batches of liposomes, with three replicates may becollected for each run.

Statistical Analysis

Means and standard deviations of release may be calculated for bothcontrol liposomes and the targeted ones. Pairwise comparisons may beperformed using ANOVA tests. Based on the assumption that bothpopulations having similar variances, two values may be consideredsignificantly different if p<0.05 and if F<F_(critical) (unlessotherwise stated).

Particle Size Measurements by DLS

Liposome size (e.g., the size of control and/or targetedimmunoliposomes) may be determined by DLS as described herein, to ensurethe formation of liposomes and to ensure that they are almost uniform. Apolydispersity (Pd) upper limit of 20% is generally acceptable in themeasurements. Table 3 summarizes the averages of the three batches withtheir standard deviation for both types of liposomes. As shown in Table4, radii of both liposomes (i.e. NH2 liposomes without antibody; andimmonoliposomes) fall within the range of SLVs and they are within theoptimal range for the EPR effects to take place, as previouslydiscussed.

A slight increase in radius after the attachment of trastuzumab isnoticed with the radius going from 89.54 nm to 101.10 nm for NH₂liposomes and immunoliposomes, respectively. This may be due to the sizeof trastuzumab molecules and their attachment to the distal end of theliposomes. Accordingly, this increase in radius may confirm theattachment of trastuzumab. In some instances, the immunoliposomes mayhave mean radius between 50-150 nm, 80-120 nm, or 90-110 nm.

TABLE 4 Summary of DLS Results Liposomes Radius (nm) Pd % NH2 liposomes 89.5417 ± 0.4964 11.2750 ± 1.1144 immunoliposomes 101.1032 ± 1.134517.2190 ± 2.3390

A single ANOVA analysis has been conducted between the radiuses of theNH2 liposomes and the immunoliposomes. Results in Table 5 show anextremely high value of F compared to F critical and a value for p-valuelower than the standard alpha value (0.05), indicating that the twotypes have different radiuses.

TABLE 5 Single-factor ANOVA analysis of radius measurements. Source ofVari- ation SS df MS F P-value F crit Be- 725.0768 1 725.0768 66.731758.43E−08 4.351244 tween Groups Within 217.3109 20 10.86555 Groups Total942.3877 21

Trastuzumab Attachment Confirmations

In order to confirm the attachment of the mAb Trastuzumab to liposomes,the BCA assay and Stewart assay may be used as described herein. The BCAassay may be used to determine the concentration of protein in thesample (μg/ml), while the Stewart assay may be used to determine DPPCconcentration in the sample (mg/ml). Combining the two methods, a w/wratio of protein to lipids in (μg/mg) may be obtained, thereby excludingthe effect of different concentrations alterations. The results forthree batches of liposomes may be averaged to confirm attachmentconsistency and standard deviation.

Also, the number of liposomes attached per each liposomes vesicle may becalculated. As described herein, the control liposomes are NH₂ liposomesthat are mixed with the antibody but without performing the attachmentreaction. The control liposomes may be purified using the samepurification column of that of immunoliposomes, to confirm that anyincrease in the protein level is only due to attached trastuzumab. Anydifference in protein level can be considered due to the presence ofattached Trastuzumab.

The summary of the results of the Stewart assay, the BCA assay and thecalculated protein level for each batch of liposomes produced accordingto some embodiments as described herein are shown in Tables 6-8,respectively. Additionally, FIG. 4 illustrates protein concentrationsper mg lipids for control and immunliposomes in each of the threebatches. In FIG. 4, the increase in the protein level forimmunoliposomes is shown, confirming the antibody attachment toimmunoliposomes. The consistency of results can also be observed frombatch to batch, with w/w protein to lipids ratio is about 1:48 inimmunoliposomes.

Also, as shown in Tables 5-7, both the control liposomes andimmunoliposomes may have approximately the same lipids concentration,which is expected because they were both made following the sameprocedure except instead of adding cyanuric chloride with acetone.

Additionally, the protein level increase was found to be a criticalfunction of the cyanuric chloride added, in which can be shown for thelast batch where a slight increase of about 2 μl resulted in slightlyhigher protein amount. However, cyanuric chloride addition may becontrolled to prevent the homopolymerization of mAb-mAb or thepossibility of the liposome-liposome attachment.

TABLE 6 Trastuzumab attachment results summary for batch 1 μg protein/mlmg lipids/ml μg protein/mg lipids control liposomes 69.6179 2.793124.9249 immunoliposomes 121.4786 2.8431 42.7278 Trastuzumab 17.8029

TABLE 7 Trastuzumab attachment results summary for batch 2 μg protein/mlmg lipids/ml μg protein/mg lipids control liposomes 76.8071 3.265623.5199 immunoliposomes 147.4385 3.3570 43.9198 Trastuzumab 20.3999

TABLE 8 Trastuzumab attachment results summary for batch 3 μg protein/mlmg lipids/ml μg protein/mg lipids control liposomes 78.5010 3.005626.1179 immunoliposomes 172.7090 3.3888 50.9647 Trastuzumab 24.8468

For the Stewart assay for measurements of lipids concentrations, lipidssolutions absorb light at 485 nm as described herein, and a calibrationcurve of known concentrations of DPPC in mg/ml versus absorbed spectrawas constructed. Samples were compared against that curve, and eachspectra value was converted to a concentration. Six replicates per batchwere used for both types of liposomes. This calibration curve was linearwhich allowed for more accuracy and flexibility in calculations.

Similar procedure was adopted when running the BCA assay, where acalibration curve was first constructed for known trastuzumab solutionconcentrations. Then each sample spectra was compared against that curveto a yield the protein concentration in μg/ml. The protein sources inthe samples were the amino acids in the antibody and the peptide pondsin the liposomes themselves. To account for that difference, proteinconcentrations for the control liposomes were measured as well.Therefore, the difference in protein amounts could be attributed only tothe amino acids in the attached trastuzumab. This is feasible because ofthe linearity of the calibration curve, where the protein concentrationcan be additive and subtractive. Additionally, the nature of the controlliposomes accounted for the calculation of attached mAb only. Some ofthe subtracted protein was due to the presence of free trastuzumab inthat sample, leaving only the effect of protein coming from attachedtrastuzumab.

Assuming a liposome size of 100 nm in radius and an average area for asingle phospholipid molecule of about 75 A°, the average number of lipidmolecules constructing a single liposomes vesicle may be 80,000. Knowingthe concentrations of the lipids and trastuzumab and their molecularweights, it was found that almost 9 trastuzumab molecules wereconjugated per liposome vesicle. This is considered to fall within theoptimal range that may induce sufficient cell cytotoxicity. In someembodiments, each immunoliposome may have about 1, 2, 4, 6, 7, 8, 10,11, 12, 14, 18, 24, 30, 50, or more than 50 trastuzumab moleculesconjugated to the liposome vesicle.

Mechanical Index Calculations

Mechanical index is a measurement of likelihood of cavitation and damageto cells and tissues, as described herein. At different mechanicalindexes, different effects may start to happen. For example, forcollapse cavitation to start, the mechanical index has to reach 0.3, andfor biological effects, it has to reach 0.6. Tissue damage begins at amechanical index of 1, where the limits set by the Food and DrugAdministration (FDA) is MI=1.9. The following formula is used tocalculate the MI.

$\begin{matrix}{{M\; I} = \frac{p^{-}\lbrack{Mpa}\rbrack}{\sqrt{f\lbrack{MHz}\rbrack}}} & {{Eq}.\mspace{14mu} \left( {2a} \right)}\end{matrix}$

Where P⁻ is the negative pressure in [Mpa], and f is frequency in [MHz].

The negative pressure can be calculated using the acoustic impedance ofwater which is 1.48 (MPa·s/m) and the intensity. This is shown inequation below.

P=√{square root over (2lz)} [Pa]  Eq. (2b)

Where l is power density in power density in W/m² and

is acoustic impedance of water in [Pa·s/m].

It is well noticed that the power densities and frequencies calculatedcorrespond to mechanical indexes of 0.11, 0.12, and 0.16 for powerdensities of 7.46, 9.85, and 17.31 (mW/cm2) respectively. In certainembodiments, the power density may be at least about: 2, 4, 6, 10, 14,18, 20, 24 30, 40, 50, or more than 50 (mW/cm2). These values are wellbelow the safe limits of any biological effects. It can be concludedthat higher power densities, up to 60 mW/cm² which corresponds to amechanical index of 0.3, can be safely implemented when using afrequency of 20 kHz ultrasound.

Low-Frequency Ultrasound (LFUS) Release Studies

As described herein, the ultrasound release experiments may be conductedat 20 kHz, and at three power densities, 7.46, 9.85, and 17.31 mW/cm².Pulsed ultrasound at 20 seconds on and 10 seconds off may be used for atotal duration of 6.3 minutes, corresponding to an actual duration of4.2 minutes. In some embodiments, the ultrasound may be pulsed for atotal duration of at least about 2 minutes, 4 minutes, 6 minutes, 10minutes, 14 minutes, 18 minutes, 20 minutes, or more than 24 minutes. Ascalcein is released, an increase in fluorescence level should beobserved. The baseline at before sonication may be measured for 60seconds before pulsed sonication was initiated. Then sonication may beapplied until a plateau was reached. After that, a sharp increase in thefluorescence level may be noticed when liposomes were lysed to spill alltheir contents. Finally, the data may be normalized using equation (1).For both types of liposomes (the control and immunoliposomes), threebatches may be used, with three replicates measurements for each batch.

The control liposomes used in the LFUS studies may be the NH₂ liposomesbut the buffer may be lowered from pH 8.5 to pH 7.4. Buffer changing maybe done, for example using the purification column. Immunoliposomes maygo through the conjugation process before changing the buffer to pH 7.4.

Low-Frequency Ultrasound Release Studies for NH₂ Liposomes

FIG. 5 illustrates the average cumulative fraction release data (CFR)for the three batches, for each power density. NH₂ liposomes calceinrelease at LFUS and relatively low intensities may be established, asshown in FIG. 5. This may be beneficial, as LFUS can be used topenetrate further into the human body than HFUS. Also, high-intensityultrasound can cause unwanted effects such as hyperthermia.

As shown in FIG. 5, the release rate may become steeper as the powerdensity increases; this is expected due to the increase in cavitationevents as the power density increases. Also, NH₂ liposomes may releasemost of all their contents (86.35%) within 3 minutes, which demonstratetheir sonosensitivity. In conclusion, the release rate may be dependenton both the power density and exposure time.

Also, as shown in FIG. 5, as sonication stops, the release may alsocease with no or substantially no delay, and any events occurring uponsonication may disappear immediately. This may support the occurrence ofstable cavitation (mechanical effects), rather than thermal effects atLFUS, since low-intensity ultrasound was used and no increase intemperatures was measured. The possible mechanism can be that LFUScauses pore-like defects in the liposomes membrane upon exposure, whichthen heals immediately in off periods.

As shown in FIG. 5, the release may be accumulative, such that the drugcan be released continuously, or pulsed, resulting in the same CFR(after the same exposure time). This may be important for the control ofthe hyperthermia effects upon the continuous exposure to ultrasound.

As summarized in Table 9, in one embodiment, NH₂ liposomes releasedalmost 86.35% of their content after 140 seconds (7 pulses) of actualsonication at 7.46 (mW/cm2), 100 seconds (5 pulses) at 9.85 (mW/cm2),and 60 seconds (3 pulses) at 17.31 (mW/cm2). It has been reported thatliposomes with no sonication may release about 3% of their contents in 3minutes. Higher release rates may be attributed to more cavitationevents. Table 9 also shows the range of ultrasound parameters that canbe selected to achieve a specific release rate.

TABLE 9 Release data summary of NH2 liposomes showing total release CFRat the plateau Power density CFR at Pulses to reach Time to reachplateau (mW/cm2) Plateau plateau (seconds) 7.46 0.8530 7 140 9.85 0.88205 100 17.31 0.8554 3 60 average 0.8635

FIG. 6 illustrates CFR measured at different pulses, and the finalplateau for NH₂ liposomes. As illustrated in FIG. 6, the CFR values mayincrease as power density increases. Also, the amount released after thethird pulse for the low power density (7.46 mW/cm²) may be almost 46% ofthe total drug encapsulated within liposomes, which can occur after 1minute of actual sonication. In some embodiments, more than 30%, 40% or50% of the total drug encapsulated may be released after the third pulsefor the low power density (7.46 mW/cm²).

FIG. 7 illustrates the fraction released for each pulse separately (notaccumulated) at 7.46 mW/cm². As illustrated, the most fraction releasedof the drug may be in the first pulse. This may be important in whichhigh initial levels of the drug are needed for biological effectiveness,and then a consequent less amounts to follow to retain that level. Insome embodiments, more than 10%, 15%, 20%, 25%, 30%, 40%, or more than50% of the total drug encapsulated by the liposome may be released afterthe first pulse. As illustrated in FIG. 7, the plateau may happen afterthe 7^(th) pulse at which afterwards the release has very low rates. Insome embodiments, the plateau may be reached after about 2, 3, 4, 5, 6,8, or more pulses.

Low-Frequency Ultrasound Release Studies for Immunoliposomes

FIG. 8 illustrates the online release rate of calcein fromimmunoliposomes at the three power densities, averaged for the threebatches of liposomes tested according to one embodiment. As shown inFIG. 8, the almost complete release of the liposomes content (92%) mayhappened after 6 pulses (120 s), 4 pulses (80 s), and 3 pulses (60 s),for 7.46, 9.85, and 17.31 mW/cm², respectively. The release data is alsoshown in Table 10. Release upon exposure to ultrasound may beestablished similarly as described herein in relation to the control NH₂liposomes. As shown in FIG. 8, the release rate in immunoliposomes mayincrease as power densities increase. Also, release may substantiallyhappen only at exposure to ultrasound, and it may be accumulative.Release may continue for a few seconds in the off period at the highestpower density, possibly due to the fact that thermal effects started tooccur. At a power density of 17.31 mW/cm², the drug may start to getinternalized into the liposomes again after the plateau. The reasoncould be hyperthermia effects allowing the drug to diffuse back, whereliposomes membrane become fragile. Normally, ultrasound exposure maystop before reaching that stage.

TABLE 10 Release data summary of immunoliposomes showing total releaseCFR at the plateau Power density CFR at Pulses to reach Time to reachplateau (mW/cm2) Plateau plateau (seconds) 7.46 0.9109 6 120 9.85 0.92574 80 17.31 0.9246 3 60 average 0.9204

FIG. 9 illustrates CFR measured at different pulses, and the finalplateau for immunoliposomes according to one embodiment. As shown inFIG. 9, CFR clearly increase with increasing intensities. After 1minute, the amount released from immunoliposomes may be 47% at a powerdensity of 7.46 mW/cm², whereas complete release may be achieved at thatsame duration for the 17.31 mW/cm² power density. In some embodiments,more than 30%, 40% or 50% of the total drug encapsulated may be releasedafter the third pulse for the low power density (7.46 mW/cm²).

FIG. 10 shows the non-accumulative fraction of drug released at eachpulse for 7.46 mW/cm² for immunoliposomes. As illustrated, the mostfraction released of the drug may be in the first pulse. This may beimportant in which high initial levels of the drug are needed forbiological effectiveness, and then a consequent less amounts to followto retain that level. In some embodiments, more than 10%, 15%, 20%, or25% of the total drug encapsulated by the liposome may be released afterthe first pulse. The plateau is shown to happen clearly after the 6thpulse, and incremental release is very low after that. In someembodiments, the plateau may be reached after 3, 4, 5, 7, 8, or morepulses.

Comparison Between the NH₂ Liposomes and Immunoliposomes Release Rates

FIG. 11 illustrates release profiles for NH₂ liposomes andimmunoliposomes at different power densities according to oneembodiment. As shown in FIG. 11, immunoliposomes may have a steeperrelease profile than the control NH₂ liposomes, indicating itssono-sensitivity, which may be beneficial in ultrasound triggeredrelease. This could be used to reach therapeutic levels in shorterexposure times.

FIGS. 12-13 illustrate fraction released after the first and the secondpulse for NH₂ liposomes and immunoliposomes at each power density,respectively. FIG. 14 illustrates final cumulative release fraction fromNH₂ liposomes and immunoliposomes at each power density. As illustratedin FIGS. 12-14, immunoliposomes may have higher drug release amountsafter the first pulse, and higher final cumulative amount of drug thatliposomes were able to release. For example, immunoliposomes may keepless than 8% encapsulated drug within immunoliposomes, compared to 13%for NH₂ liposomes at the end of the sonication period.

Release Kinetics Models

Modeling the release kinetics may help in predicting release atdifferent conditions including lipid and the agents' composition, powerdensity and frequency. In addition, it may help for the design ofequipment to optimize release. The data presented above can be used tofind the best fitting model that can successfully represent the calceinrelease from targeted and non-targeted liposomes. Following models maybe used to find the best fitting model.

The zero-order model is derived from a basic understanding of thephysical process of tablets, and capsules where the drug is releasedvery slowly at a constant rate. Assuming that the area is constant andno equilibrium conditions are obtained, equation (3) represent themodel:

Q _(t) −Q ₀ =K ₀ t  Eq. (3)

where Q_(t) represents the amount of drug dissolved in time t; Q₀represents the initial amount of drug in the solution; and K₀ representsa zero-order release constant. Rearranging the equation to fit ournormalized data, is presented in equation (4):

CFR=k ₀ t  Eq. (4)

where CFR represents cumulative fraction released; t represents time inseconds; and k₀ represents zero-order release constant in percentage persecond. A plot of CFR against time will deliver the release constant asthe slope.

A first-order model is based on assumption that dissolution includes asurface action. It can be used accurately to model the release ofwater-soluble drugs in porous matrices. It is mathematically representedby equation (5):

$\begin{matrix}{\frac{d\; c}{dt} = {{- k_{1}}C}} & {{Eq}.\mspace{14mu} (5)}\end{matrix}$

where C represents drug concentration at time t; and k₁ representsfirst-order rate constant in seconds⁻¹. After integrating andrearranging to make it compatible with the data taken, it yieldsequation (6):

log CFR=k ₁ t+constant  Eq. (6)

Accordingly, a plot of log(CFR) versus time will yield the slope k₁.

Higuchi model describes release as a diffusion based process.Applications of the model include transdermal systems and matrix tabletscarrying water-soluble drugs. The model assumes the following: 1)Diffusion is in one dimension only; 2) The concentration of the druginside the matrix is higher than its solubility; 3) The drug moleculesare much smaller than the matrix thickness; 4) Constant drugdiffusivity; 5) The matrix change in dimensions is negligible; 6)Perfect sink conditions in the release environment.

The simplified equation for the Higuchi model can be seen in equation(7):

Q=k _(H)√{square root over (t)}  Eq. (7)

where k_(H) represents Higuchi release constant and Q represents amountof drug released in time t. Rearranging to convert Q to CFR yieldsequation (8), that can be used to obtain Higuchi release constant uponplotting CFR versus square root of time.

CFR=k _(H) √{square root over (t)}  Eq. (8)

Korsmeyer-Peppas model is a simple model describing the release fromporous hydrophilic polymers. Korsmeyer-Peppas or the power-law as it'ssometimes called is a more general model than Higuchi. It takes intoaccount the effects of swelling and dissolution, and it does not assumea diffusion-based release. At small t, the model can be simply shown asin equation (9):

$\begin{matrix}{\frac{M_{t}}{M_{\infty}} = {k_{kp}t^{n}}} & {{Eq}.\mspace{14mu} (9)}\end{matrix}$

where

$\frac{M_{t}}{M_{\infty}}$

represents the fraction of drug released at time t; k_(kp) representsthe Korsmeyer-Peppas release rate constant; and n represents the releaseexponent. The rate constant changes with different shapes andstructures. The exponent value indicates the mechanism of the release.For the case of cylindrical shapes, if n≤0.45, then the release followsFick's law (diffusion-dependent), if 0.45<n<0.89 then the release isnon-Fickian, if n=0.89 the release follows relaxation transport, and ifn>0.89 then the release is considered super case transport. This is whythis model is used to study the release when the mechanisms are notknown.

After adjusting the model to our type of data and linearizing, equation(10) is realized, and a plot of the log(CFR) versus log(t) can yieldlog(k_(KP)) as the intersect and the n-value as a slope.

log(CFR)=log(k _(kp))+n log(t)  Eq. (10)

Hixson-Crowell model is based on the proportionality of the sphereregular area to the square root of its volume. The relation can be seenin equation (11).

W ₀ ^(1/3) −W _(t) ^(1/3) =k _(HC) t  Eq. (11)

where W₀ represents the amount of drug initially inside the liposome;W_(t) represents the remaining amount of drug in the liposomes; andk_(HC) represents the proportionality constant.

Rearranging the resultant equation to get CFR in one side, yieldsequation (12).

(1−CFR)^(1/3)=1−k _(HC) ^(t)  Eq. (12)

This model assumes that release is controlled by the dissolution of thedrug particles and not their diffusion through the pores of the matrix.It also takes into account the reduction in the particle size as itdissolved in the solution. Plotting 1−(1−CFR)^(1/3) versus time shouldyield a straight line with −k_(HC) as the slope after setting theintercept at 1.

In embodiments, the Baker-Lonsdale model describes the release fromspherical matrices by developing the Higuchi model, and the expressionis shown in equation (13). The resultant equation when converting therelease in terms of CFR is described in equation (14).

$\begin{matrix}{{{\frac{3}{2}\left\lbrack {1 - \left( {1 - \frac{M_{t}}{M_{\infty}}} \right)^{\frac{2}{3}}} \right\rbrack} - \frac{M_{t}}{M_{\infty}}} = {k_{BL}t}} & {{Eq}.\mspace{14mu} (13)}\end{matrix}$

where M_(t) represents the drug release amount at time t; M_(∞)represents the total amount released at infinite time (initial amountinside the liposomes); and k_(BL) represents the release constant.

3/2[1−(1−CFR)^(2/3)]−CFR=k _(BL) t  Eq. (14)

Plotting the left hand equation (14) versus time will result in astraight line with k_(BL) as the slope.

The Weibull model is a general empirical relation that describesdifferent dissolution rates of matrix type systems. The relation isshown in equation (15) and the altered form to incorporate our data isshown in equation (16), whereas the linearized form can be seen inequation (17).

$\begin{matrix}{m = {1 - e^{\lbrack\frac{- {({t - T})}^{b}}{a}\rbrack}}} & {{Eq}.\mspace{14mu} (15)}\end{matrix}$

Where m represents accumulated fraction of the drug; a represents ascale parameter that describes time dependence; b describes shape ofdissolution curve; and T accounts for the time lag in dissolutionprocess (taken=0).

$\begin{matrix}{{1 - {C\; F\; R}} = e^{\lbrack\frac{- {(t)}^{b}}{a}\rbrack}} & {{Eq}.\mspace{14mu} (16)} \\{{\log \left( {- {\ln \left( {1 - {C\; F\; R}} \right)}} \right)} = {{b\; {\log (t)}} + {\log \; k_{w}}}} & {{Eq}.\mspace{14mu} (17)}\end{matrix}$

Where k_(w) is

$\frac{1}{a},$

and represents Weilbull rate constant. Plotting the expression ofequation (17) versus log(t) will yield b as the slope and log(k_(w)) asthe intersection.

Hopfenberg model assumes that the surface remains constant duringeroding. It also assumes that a zero-order mechanism will take placethroughout the eroding process, whether the drug was loaded chemically(attached), or physically (dissolved, dispersed). The model considersthe diffusion process to be so rapid that it cannot be rate determining.The cumulative fraction released at time t is described by the model inequation (18).

$\begin{matrix}{\frac{M_{t}}{M_{\infty}} = {1 - \left\lbrack {1 - \frac{k_{0}t}{C_{L}a}} \right\rbrack^{n}}} & {{Eq}.\mspace{14mu} (18)}\end{matrix}$

Where

$\frac{M_{t}}{M_{\infty}}$

represents cumulative fraction released; k₀ represents the zero orderrate constant describing the eroding; C_(L) represents the initial drugloading; a represents the system's half thickness (radius for a sphere);and n represents 1, 2, or 3, for a slap, cylinder, and a sphererespectively. Rearranging yields equation (19) in terms of CFR, which issimilar to the Hixson-Crowell equation after rearranging. This is notsurprising since some relations can reduce to others in special cases.Plotting the left-hande against time will result in a straight line of alope k_(HF).

1−(1−CFR)^(1/3) =k _(Hf) t  Eq. (19)

Where

$\begin{matrix}{k_{Hf} = \frac{k_{0}}{C_{L}a}} & {{Eq}.\mspace{14mu} (19)}\end{matrix}$

Gompertz model is for drugs with good solubility and intermediaterelease. The relation is exponential, and is shown in equation (20). Themodel has a sharp increase, which then converges gradually to a plateau.

$\begin{matrix}{\frac{X_{t}}{X_{\max}} = e^{{- \alpha}\; e^{\beta \; \log \; t}}} & {{Eq}.\mspace{14mu} (20)}\end{matrix}$

Where X_(t) represents fraction dissolved at time t; X_(max) representsmaximum dissolution; α represents scale parameter describingnon-dissolved portion at time t=1; and β represents dissolution rate perunit time. Rearranging assuming X_(max)=1 is shown in equation (20).

ln(−ln CFR)=k _(G) log t+ln α  Eq. (21)

Where k_(G)=p

According to equation (21), plotting the left-hand versus log(t) willresult in a straight line with k_(G) as the slop and ln α as theintersection.

Kinetics Models Accuracy for NH₂ Liposomes

Cumulative fraction release of each batch of NH₂ liposomes describedherein may be used in equations (4), (6), (8), (10), (12), (14), (17),(19), and (21) to construct the following graphs. To test modelsrepresentation of the drug release kinetics, a straight line shouldappear in the graph, if the model was accurate. The suitability of eachmodel may be determined upon creating a straight trendline of the plot.Then to test how close the data points are to this straight line, R² maybe calculated. Generally, R² is an indication of the model capability toaccurately represent the release data. Additionally, Parity plots werealso used to demonstrate the best fitting model. FIGS. 15-23 wereillustrated using the data from batch 1 of NH₂ liposomes at a powerdensity of 7.46 (mW/cm2), where each R² is shown on each of FIGS. 15-23.FIG. 15 illustrates zero-order plot for NH₂ liposomes, batch #1. FIG. 16illustrates first-order plot for NH₂ liposomes, batch #1. FIG. 17illustrates Higuchi model for NH₂ liposomes, batch #1. FIG. 18illustrates Korsmeyer-Peppas model for NH₂ liposomes, batch #1. FIG. 19illustrates Hixson-Crowell model for NH₂ liposomes, batch #1. FIG. 20illustrates Baker-Lonsdale model for NH₂ liposomes, batch #1. FIG. 21illustrates Weibull model for NH₂ liposomes, batch #1. FIG. 22illustrates Hopfenberg model for NH₂ liposomes, batch #1. FIG. 23illustrates Gompertz model for NH₂ liposomes, batch #1.

The R² values of the models for all the NH₂ liposomes batches at eachpower density according to one embodiment are summarized in Tables 11and 12, in which averages of each model fitting parameter (R²) is shownat the bottom of the table. It can be seen that Korsmeyer-Peppas showsthe highest R² averaged values for NH₂ liposomes (0.9952), meaning thatit is the best model to fit the release data. This can also bevisualized in the parity plots in FIGS. 24-26 plotted for the average ofthe three batches of NH₂ liposomes at each power density, usingparameters estimated for each model fit. As illustrated in FIGS. 24-26,Hixson-Crowell and Hopfenberg (colored overlapping as grey) are thesecond closest models after Korsmeyer-Peppas (colored in black) to theactual model (colored in red). Fourth closest was the Weibull model,while the rest of the models failed to accurately represent the releasedata.

TABLE 11 R² values of different models for NH2 liposomes at each powerdensity NH2 R² liposomes Zero First Korsmeyer- values batch order orderHiguchi Peppas 7.46 1 0.9490 0.7697 0.9334 0.9922 (mW/cm2) 2 0.93190.7451 0.9434 0.9951 3 0.9509 0.7697 0.9325 0.9944 9.85 1 0.9688 0.78110.9163 0.9961 (mW/cm2) 2 0.9558 0.7658 0.9280 0.9953 3 0.9618 0.78210.9224 0.9952 17.31  1 0.9718 0.7928 0.9094 0.9950 (mW/cm2) 2 0.95670.7642 0.9282 0.9973 3 0.9724 0.7823 0.9089 0.9965 Average 0.9577 0.77250.9247 0.9952

TABLE 12 R² values of different models for NH2 liposomes at each powerdensity NH2 liposomes Hixson- R² values batch Crowell Baker-LonsdaleWeibull Hopfenberg Gompertz 7.46 1 0.9954 0.9008 0.9847 0.9954 0.8808(mW/cm2) 2 0.9954 0.9117 0.9899 0.9952 0.879 3 0.9934 0.8752 0.98320.9934 0.8566 9.85 1 0.9957 0.8718 0.9868 0.9958 0.8744 (mW/cm2) 20.9967 0.8853 0.987 0.9967 0.8689 3 0.992 0.8606 0.9831 0.9920 0.857117.31 1 0.9839 0.8251 0.9801 0.9839 0.8422 (mW/cm2) 2 0.9932 0.86620.9878 0.9932 0.8576 3 0.9822 0.8212 0.9815 0.9822 0.8333 Average 0.99200.8687 0.9849 0.9920 0.8611

Models Accuracy for Immunoliposomes

The same analysis implemented for NH₂ liposome may be conducted also forimmunoliposomes. The summary for the R² values according to oneembodiment are presented in Tables 13 and 14, in which Korsmeyer-Peppasdemonstrated to have the highest R² averaged value followed byzero-order, and Weibull models. This can also be visualized in theparity plot illustrated in FIGS. 27-29, where black coloredKorsmeyer-Peppas model was the closest to the red colored actual CFRvalues, followed by the green colored Weibull model, and then the bluecolored zero-order model, and finally the Hixson-Crowell and Hopfenbergmodels overlapping as grey color.

As described herein, both types of liposomes (i.e. the control NH₂liposomes and immunoliposomes) may have similar behavior andconsequently similar mechanisms. The Korsmeyer-Peppas model revealed theapparent diffusion release mechanism by calculating the n value in themodel. The n values were found to be 0.7742 and 0.7896 for NH₂ liposomesand immunoliposomes respectively. This could be averaged for both as0.7819. This value falls in the non-Fickian transport upper limit, andclose to the super transport region. The adherence of the data toKorsmeyer-Peppas, Hopfenberg, and Hixson-Crowell, assumediffusion-driven and dissolution-driven mechanism. This could beunderstood considering that ultrasound make pore-like deformationsduring sonication allowing the drug to diffuse easily and rapidly to theouter environment.

TABLE 13 R² values of different models for immunoliposomes at each powerdensity Immu- R² noliposomes Zero First Korsmeyer- values batch orderorder Higuchi Peppas 7.46 1 0.9729 0.7991 0.9027 0.9937 (mW/cm2) 20.9784 0.7924 0.9050 0.9936 3 0.9838 0.8135 0.8856 0.9915 9.85 1 0.98120.8136 0.8782 0.9919 (mW/cm2) 2 0.9892 0.8221 0.8745 0.9906 3 0.98920.8431 0.8528 0.9847 17.31  1 0.9820 0.8160 0.8560 0.9936 (mW/cm2) 20.9905 0.8066 0.8642 0.9957 3 0.9886 0.8399 0.8640 0.9887 Average 0.98400.8163 0.8759 0.9916

TABLE 14 R² values of different models for immunoliposomes at each powerdensity Immunoliposomes Hixson- Baker- R² values batch Crowell LonsdaleWeibull Hopfenberg Gompertz 7.46 1 0.9509 0.7587 0.9616 0.9509 0.7554(mW/cm2) 2 0.9784 0.8116 0.9712 0.9784 0.7991 3 0.956 0.7667 0.95820.956 0.7599 9.85 1 0.9379 0.7209 0.9612 0.9379 0.7611 (mW/cm2) 2 0.94330.7386 0.9557 0.9433 0.7472 3 0.9066 0.7044 0.9276 0.9066 0.6735 17.31 10.8851 0.6641 0.9409 0.8851 0.6675 (mW/cm2) 2 0.9208 0.7231 0.95170.9208 0.702 3 0.9186 0.7121 0.9379 0.9186 0.6963 Average 0.9331 0.73340.9518 0.9331 0.7291

Calculations of k_(KP) Values

According to the Korsmeyer-Peppas model, a log-inverse of the intersectvalue yields the k_(kp) rate constant. Table 15 illustrates rateconstant of Korsmeyer-Peppas model for both types of liposomes at eachpower density.

TABLE 15 Rate constant of Korsmeyer-Peppas model for both types ofliposomes at each power density NH2 liposomes 7.46 (mW/cm²) 9.85(mW/cm²) 17.31 (mW/cm²) Kkp values (n = 0.7742) Batch # 1 1.9561E−022.3243E−02 3.1710E−02 Batch # 2 2.1592E−02 2.6303E−02 3.4135E−02 Batch #3 2.1404E−02 2.6934E−02 3.1261E−02 Average 2.0853E−02 2.5493E−023.2369E−02 Std. dev  1.12E−03  1.97E−03  1.55E−03 Immunoliposomes (n =0.7896) Batch # 1 2.2594E−02 2.5627E−02 3.2352E−02 Batch # 2 1.9770E−022.5322E−02 3.0409E−02 Batch # 3 2.0455E−02 2.7277E−02 3.5067E−02 Average2.0940E−02 2.6075E−02 3.2609E−02 Std. dev  1.47E−03  1.05E−03  2.34E−03

In Table 16, a two-factor ANOVA analysis is shown. As shown in Table 15,the F-value is lower than the F-critical value, and hence no significantdifference exists between k_(kp) values for both types of liposomes.This indicates that the release rate constant may not be affected by thetype of liposomes (NH₂ liposomes or immunoliposomes). This means thatimmunoliposomes follow the same release pattern as the control ones. Thesecond F-value is shown to be higher that the F-critical, whichindicates that k_(kp) are significantly affected by the power density.This indicates that a higher release rate may be obtained with increasedpower density.

TABLE 16 Two-factor ANOVA analysis of KKP values Source of Variation SSdf MS F p-value F crit Sample 4.14E−07 1 4.1383E−07 0.1524 0.7031 4.7472Columns 0.000406 2 0.00020324 74.822 2E−07 3.8853 Interaction 1.93E−07 29.6343E−08 0.0355 0.9653 3.8853 Within 3.26E−05 12 2.7163E−06 Total0.00044 17

OTHER EMBODIMENTS

The foregoing description and examples has been set forth merely toillustrate the disclosure and are not intended as being limiting. Eachof the disclosed aspects and embodiments of the present disclosure maybe considered individually or in combination with other aspects,embodiments, and variations of the disclosure. In addition, unlessotherwise specified, none of the steps of the methods of the presentdisclosure are confined to any particular order of performance.Modifications of the disclosed embodiments incorporating the spirit andsubstance of the disclosure may occur to persons skilled in the art andsuch modifications are within the scope of the present disclosure.Furthermore, all references cited herein are incorporated by referencein their entirety.

Terms of orientation used herein, such as “top,” “bottom,” “horizontal,”“vertical,” “longitudinal,” “lateral,” and “end” are used in the contextof the illustrated embodiment. However, the present disclosure shouldnot be limited to the illustrated orientation. Indeed, otherorientations are possible and are within the scope of this disclosure.Terms relating to circular shapes as used herein, such as diameter orradius, should be understood not to require perfect circular structures,but rather should be applied to any suitable structure with across-sectional region that can be measured from side-to-side. Termsrelating to shapes generally, such as “circular” or “cylindrical” or“semi-circular” or “semi-cylindrical” or any related or similar terms,are not required to conform strictly to the mathematical definitions ofcircles or cylinders or other structures, but can encompass structuresthat are reasonably close approximations.

Conditional language used herein, such as, among others, “can,” “might,”“may,” “e.g.,” and the like, unless specifically stated otherwise, orotherwise understood within the context as used, is generally intendedto convey that some embodiments include, while other embodiments do notinclude, certain features, elements, and/or states. Thus, suchconditional language is not generally intended to imply that features,elements, blocks, and/or states are in any way required for one or moreembodiments or that one or more embodiments necessarily include logicfor deciding, with or without author input or prompting, whether thesefeatures, elements and/or states are included or are to be performed inany particular embodiment.

Conjunctive language, such as the phrase “at least one of X, Y, and Z,”unless specifically stated otherwise, is otherwise understood with thecontext as used in general to convey that an item, term, etc. may beeither X, Y, or Z. Thus, such conjunctive language is not generallyintended to imply that certain embodiments require the presence of atleast one of X, at least one of Y, and at least one of Z.

The terms “approximately,” “about,” and “substantially” as used hereinrepresent an amount close to the stated amount that still performs adesired function or achieves a desired result. For example, in someembodiments, as the context may dictate, the terms “approximately”,“about”, and “substantially” may refer to an amount that is within lessthan or equal to 10% of the stated amount. The term “generally” as usedherein represents a value, amount, or characteristic that predominantlyincludes or tends toward a particular value, amount, or characteristic.As an example, in certain embodiments, as the context may dictate, theterm “generally parallel” can refer to something that departs fromexactly parallel by less than or equal to 20 degrees.

Unless otherwise explicitly stated, articles such as “a” or “an” shouldgenerally be interpreted to include one or more described items.Accordingly, phrases such as “a device configured to” are intended toinclude one or more recited devices. Such one or more recited devicescan be collectively configured to carry out the stated recitations. Forexample, “a processor configured to carry out recitations A, B, and C”can include a first processor configured to carry out recitation Aworking in conjunction with a second processor configured to carry outrecitations B and C.

The terms “comprising,” “including,” “having,” and the like aresynonymous and are used inclusively, in an open-ended fashion, and donot exclude additional elements, features, acts, operations, and soforth. Likewise, the terms “some,” “certain,” and the like aresynonymous and are used in an open-ended fashion. Also, the term “or” isused in its inclusive sense (and not in its exclusive sense) so thatwhen used, for example, to connect a list of elements, the term “or”means one, some, or all of the elements in the list.

Overall, the language of the claims is to be interpreted broadly basedon the language employed in the claims. The language of the claims isnot to be limited to the non-exclusive embodiments and examples that areillustrated and described in this disclosure, or that are discussedduring the prosecution of the application.

Although systems and methods for and of making liposomes, includingcontrol and targeted liposomes, have been disclosed in the context ofcertain embodiments and examples, this disclosure extends beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses of the embodiments and certain modifications and equivalentsthereof. Various features and aspects of the disclosed embodiments canbe combined with or substituted for one another in order to form varyingmodes of systems and methods for and of making liposomes, includingcontrol and targeted liposomes. The scope of this disclosure should notbe limited by the particular disclosed embodiments described herein.

Certain features that are described in this disclosure in the context ofseparate implementations can be implemented in combination in a singleimplementation. Conversely, various features that are described in thecontext of a single implementation can be implemented in multipleimplementations separately or in any suitable subcombination. Althoughfeatures may be described herein as acting in certain combinations, oneor more features from a claimed combination can, in some cases, beexcised from the combination, and the combination may be claimed as anysubcombination or variation of any subcombination.

While the methods and devices described herein may be susceptible tovarious modifications and alternative forms, specific examples thereofhave been shown in the drawings and are herein described in detail. Itshould be understood, however, that the invention is not to be limitedto the particular forms or methods disclosed, but, to the contrary, theinvention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the various embodiments describedand the appended claims. Further, the disclosure herein of anyparticular feature, aspect, method, property, characteristic, quality,attribute, element, or the like in connection with an embodiment can beused in all other embodiments set forth herein. Any methods disclosedherein need not be performed in the order recited. Depending on theembodiment, one or more acts, events, or functions of any of thealgorithms, methods, or processes described herein can be performed in adifferent sequence, can be added, merged, or left out altogether (e.g.,not all described acts or events are necessary for the practice of thealgorithm). In some embodiments, acts or events can be performedconcurrently, e.g., through multi-threaded processing, interruptprocessing, or multiple processors or processor cores or on otherparallel architectures, rather than sequentially. Further, no element,feature, block, or step, or group of elements, features, blocks, orsteps, are necessary or indispensable to each embodiment. Additionally,all possible combinations, subcombinations, and rearrangements ofsystems, methods, features, elements, modules, blocks, and so forth arewithin the scope of this disclosure. The use of sequential, ortime-ordered language, such as “then,” “next,” “after,” “subsequently,”and the like, unless specifically stated otherwise, or otherwiseunderstood within the context as used, is generally intended tofacilitate the flow of the text and is not intended to limit thesequence of operations performed. Thus, some embodiments may beperformed using the sequence of operations described herein, while otherembodiments may be performed following a different sequence ofoperations.

Moreover, while operations may be depicted in the drawings or describedin the specification in a particular order, such operations need not beperformed in the particular order shown or in sequential order, and alloperations need not be performed, to achieve the desirable results.Other operations that are not depicted or described can be incorporatedin the example methods and processes. For example, one or moreadditional operations can be performed before, after, simultaneously, orbetween any of the described operations. Further, the operations may berearranged or reordered in other implementations. Also, the separationof various system components in the implementations described hereinshould not be understood as requiring such separation in allimplementations, and it should be understood that the describedcomponents and systems can generally be integrated together in a singleproduct or packaged into multiple products. Additionally, otherimplementations are within the scope of this disclosure.

Some embodiments have been described in connection with the accompanyingfigures. Certain figures are drawn and/or shown to scale, but such scaleshould not be limiting, since dimensions and proportions other than whatare shown are contemplated and are within the scope of the embodimentsdisclosed herein. Distances, angles, etc. are merely illustrative and donot necessarily bear an exact relationship to actual dimensions andlayout of the devices illustrated. Components can be added, removed,and/or rearranged.

Further, the disclosure herein of any particular feature, aspect,method, property, characteristic, quality, attribute, element, or thelike in connection with various embodiments can be used in all otherembodiments set forth herein. Additionally, any methods described hereinmay be practiced using any device suitable for performing the recitedsteps.

The methods disclosed herein may include certain actions taken by apractitioner; however, the methods can also include any third-partyinstruction of those actions, either expressly or by implication. Forexample, actions such as “positioning an electrode” include “instructingpositioning of an electrode.”

In summary, various embodiments and examples of systems and methods forand of making liposomes, including control and targeted liposomes, havebeen disclosed. Although the systems and methods for and of makingliposomes, including control and targeted liposomes, have been disclosedin the context of those embodiments and examples, this disclosureextends beyond the specifically disclosed embodiments to otheralternative embodiments and/or other uses of the embodiments, as well asto certain modifications and equivalents thereof. This disclosureexpressly contemplates that various features and aspects of thedisclosed embodiments can be combined with, or substituted for, oneanother. Thus, the scope of this disclosure should not be limited by theparticular disclosed embodiments described herein, but should bedetermined only by a fair reading of the claims that follow.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited. Numbers preceded by a term such as “about” or“approximately” include the recited numbers and should be interpretedbased on the circumstances (e.g., as accurate as reasonably possibleunder the circumstances, for example ±5%, ±10%, ±15%, etc.). Forexample, “about 1 V” includes “1 V.” Phrases preceded by a term such as“substantially” include the recited phrase and should be interpretedbased on the circumstances (e.g., as much as reasonably possible underthe circumstances). For example, “substantially perpendicular” includes“perpendicular.” Unless stated otherwise, all measurements are atstandard conditions including temperature and pressure.

What is claimed is:
 1. A method of treating breast cancer in a mammal,the method comprising: delivering an actively targeted liposome to themammal, the actively targeted liposome comprising: a lipid bilayerforming a spherical shell, wherein the spherical shell comprises aninterior liposomal cavity, a plurality of trastuzumab molecules linkedto a surface of the actively targeted liposome, and a chemotherapeuticdrug, the chemotherapeutic drug comprising at least one of a hydrophilicchemotherapeutic drug contained within the interior liposomal cavity anda hydrophobic chemotherapeutic drug contained within the lipid bilayerof the actively targeted liposome; and allowing the actively targetedliposome to circulate throughout a circulatory system of the mammal fora time sufficient to allow aggregation of a therapeutic quantity ofactively targeted liposomes at a treatment area comprising a breastcancer; and applying ultrasound to the treatment area such that theactively targeted liposome is critically disrupted thereby releasing thechemotherapeutic drug in the treatment area.
 2. The method of claim 1,wherein the ultrasound applied to the treatment area comprises a lowfrequency ultrasound.
 3. The method of claim 2, wherein the lowfrequency ultrasound comprises a 20 kHz with a power density of one of7.46 W/cm², 9.85 W/cm², and 17.31 W/cm².
 4. The method of claim 2,wherein the low frequency ultrasound applied to the treatment area isapplied for less than about 6 minutes.
 5. The method of claim 1, whereinthe ultrasound applied to the treatment area comprises high frequencyultrasound.
 6. The method of claim 1, wherein pulsed ultrasound isapplied to the treatment area.
 7. The method of claim 1, wherein thelipid bilayer of the actively targeted liposome comprises one or morePEGylated lipids.
 8. The method of claim 7, wherein the plurality oftrastuzumab molecules are linked to a distal end of the PEG chain. 9.The method of claim 1, wherein the actively targeted liposome comprisesa protein density of 7.5-30 molecules per liposome.
 10. The method ofclaim 1, wherein the actively targeted liposome comprises a mean radiusbetween 50-150 nm.
 11. The method of claim 1, wherein the activelytargeted liposome comprises 6 to 12 trastuzumab molecules.
 12. Themethod of claim 6, wherein the ultrasound is pulsed 2 times.
 13. Themethod of claim 6, wherein the ultrasound is pulsed 3 times.
 14. Themethod of claim 1, wherein the chemotherapeutic drug comprises calcein.15. The method of claim 1, wherein the chemotherapeutic drug is selectedfrom the group of doxorubicin, annamycin, daunorubicin, vincristine,cisplatin derivatives, paclitaxel 5-fluorouracil derivatives,camptothecin derivatives, and retinoids.
 16. The method of claim 1,wherein the actively targeted liposome comprises a plurality ofvesicles, the plurality of vesicles linked to about 9 trastuzumabmolecules.
 17. The method of claim 1, wherein the plurality oftrastuzumab molecules are linked to the surface of the actively targetedliposome using cyanuric chloride.